細胞的生物化學

以生物化學的觀點，複習細胞的重要活動。最近生命科學的大趨勢，是以分
子層次的觀察，研究細胞乃至於器官或生物整體的生理現象，稱為 molecular cell
biology。

1.原核細胞：
原核細胞的代表 大腸菌 (E. coli)，構造較為簡單，是分子生物學的主要研究對
象。
a.細胞壁 (cell wall) 由 胜肽聚醣 (peptidoglycan) 構成，結構堅固，其功能
有︰ (1) 保護細胞； (2) 細胞內外物質及訊息的交通； (3) 抗原性及 (噬
菌体) 接受体。
b.鞭毛 (flagella) 使細菌運動，而 纖毛 (pili) 為細菌交配時的管道。
c.細胞膜 (cell membrane) 控制細胞內外的選擇性交通，膜蛋白有重要功能。
d. 細 胞 質 (cytoplasma) 散 佈 著 各 種 分 子 ， 主 要 是 可 溶 性 酵 素 、 核 糖 体
(ribosome)。
e.核區 (nuclear region) 不是真正的細胞核，散佈著遺傳物質 DNA，細菌通
常有一或數條 DNA 分子；細胞質中有環狀的 質體 DNA，是基因選殖
的主要載體。
2.古生菌：
是一種介於原核與真核細胞間的細菌。古生菌與已知的原核細胞，在生化性質上
有相當差異；喜生長在極端的條件，極類似地球演化的早期狀態；可分為三大類：
a.Methanogens： 甲烷菌極度厭氧，利用二氧化碳及氫氣產生甲烷。
b.Halophiles： 嗜鹽菌，生長在如死海的高鹽濃度區。
c.Thermacidophiles： 嗜酸熱菌，生長在火山口及溫泉帶，可耐酸至 pH 2。
3.真核細胞：
原核細胞與真核細胞的最大差異，在於後者有許多 胞器 (cellular organelles)，構
造複雜；而最顯著的一個胞器，就是 細胞核 (nucleus)，原核細胞無細胞核。
a.細胞核：由雙層核膜包圍著，膜上有 核孔，核內有 核仁 (nucleolus)，核仁
含 大 量 RNA ， 其 餘 的 核 質 (nucleoplasm) 部 分 則 散 佈 著 染 色 質
(chromatin)，染色質含遺傳物質 DNA，在細胞分裂前，染色質會凝集成 染
色体 (chromosome)。 細胞核可能是由細胞外膜向內皺縮，包住染色體後形
成球狀所造成。
b.內質網 (endoplasmic reticulum, ER)：是細胞蛋白質的合成及輸送系統，依外
形分為 RER (rough ER) 及 SER (smooth ER)； RER 在其膜上附著顆粒狀
的 核糖体 (ribosome)，蛋白質合成後可通過內質網膜分泌到細胞外；不分
泌到胞外的蛋白質，則由游離散佈在細胞質中的核醣体來製造。 SER 表面
光滑，沒有核糖体附著，可能與脂質的合成有關。
c.高爾基氏体 (Golgi body)： 是細胞內蛋白質的 集散地 與 加工場。
(1) 由內質網輸送來的蛋白質集中於此，分類後一部分分泌出細胞外。
(2) 不 分 泌 出 細 胞 的 蛋 白 質 ， 則 集 中 後 包 裝 成 小 球 体 ， 即 為 微 体
(microbodies)。
(3) 醣蛋白 (glycoprotein) 等在此修飾加上醣類。
d.微体 (microbodies)： 有很多種，都含某種劇烈的酵素，有特定的生化功能。
(1) Lysosomes (溶酶体) 含有 溶菌酶 (lysozyme) 等多種水解酵素，以消化
外來蛋白質、核酸、醣類等分子。植物細胞內的對等胞器為液泡，其体
積都很大。
(2) Peroxisomes 含有 觸酶 (catalase)，把有害細胞的 H2O2 分解成水。
(3) Glyoxosomes 可把脂質轉化成醣類，也是植物特有胞器的一種。
e.細胞骨架系統 (cytoskeleton elements)：由許多小管所交錯構成，用以支持細
胞，並行細胞運動、胞內運輸 及 細胞分裂。
f.細胞膜 (cell membrane)︰ 真核細胞最外層胞膜上附有許多蛋白質，有複雜
的功能。
(1) 細胞間辨認 的特異性標記，如免疫學的各種 T 細胞上都有不同標記。
(2) 荷爾蒙受体，與其配體分子接觸後，可引發細胞內一連串信息傳導反應。
(3) 細胞內外離子的 輸送幫浦，也都是由蛋白質所組成。
g.粒線体 (mitochondria)： 是細胞產生能量的地方。
(1) 由雙層膜組成，內層向細胞內伸展，皺褶成為 瘠 (cristae)。 瘠上有
顆粒密佈，是藉 呼吸鏈 進行能量代謝的地方，可生成 ATP。
(2) 粒線体有自己的 DNA，也可以合成蛋白質，是細胞內的自治區；可能
是可以行呼吸作用的原核細胞，侵入早期的真核細胞後，留在宿主細胞
中共生。
h.葉綠体 (chloroplast) 與 造粉体 (amyloplast)：
(1) 葉綠体進行光合作用捕捉太陽光能，與細胞壁、液泡及造粉体都是 植
物 特有胞器。葉綠體是地球生物圈最關鍵的一環，缺少葉綠體將導致
所有生物滅亡。
(2) 造粉体含有大量澱粉粒，與葉綠体都屬 胞質体 (plastid)，二者是 同源
器官，都是由相同的前體 (proplastid) 演變來，有的還可互相轉變。
(3) 胞質体也都有自己的 DNA，可能是早期的原核光合菌，進入真核細胞
後產生的共生系統。粒線體與胞質體這兩種共生胞器，都與能量的代謝
有關。
i.其它：
(1) 細胞外套 (cell coat) 只有部份動物細胞才有，會表現 抗原性； 癌細胞
的細胞外套成分可以改變，以逃避免疫系統。
(2) 微粒体 (microsome) 是細胞打碎後，內質網破片形成的人為小球，並非
胞器。
 (3) 病毒 無法歸類入任何一類生物，卻能在細胞中寄生繁衍；因病毒在各
種細胞、甚至物種間游走，夾帶部分染色體片段，可能對演化有所影響。
對人體而言，病毒可刺激免疫系統，也許不全都是負面的影響。
4.細胞的組成分子：
生物体內許多重要的巨分子，都是由單位小分子所組成。古典生化注重上述
分子的化學反應以及生理代謝，近代生化則以核酸、蛋白質及酵素為研究中心，
現代則深入分子生物學層次，探討 基因 及其 調節機制。
a.生物分子依其大小，可分為小分子及巨分子 (macromolecule)，巨分子是由小
分子的 單元体 (monomer) 為堆積單位，一個個接起來。例如蛋白質是由胺
基酸所組成的。
b.常見的小分子有胺基酸、單醣、脂質、核苷酸等，都是体內分子的 運輸 形
式； 而大分子有蛋白質、多醣、核酸等，是 功能、構造 或 貯藏 形式。 另
有許多具有生物活性的小分子，如輔酶及維生素，其中以水含量最多，作用
也最廣泛。
c.巨分子的 序列 是極為重要的，核酸的序列藏著遺傳信息，蛋白質的序列是
取決於核酸的序列，而蛋白質的序列決定其構造與生理功能。 因此，在巨
分子的世界裡，序列幾乎決定一切。
5.分子間的作用力：
分子與分子之間，或者同一分子裡面，有多種非共價的作用力存在，可使得
分子間相吸的是引力，互相排斥的為斥力。 這些微弱作用力是構成 分子構形
(conformation) 及 分 子 間 親 和 力 (affinity) 的 主 要 因 素 ， 統 稱 為 二 級 鍵
(secondary bonds)。
a.離子鍵 (electrostatic bond) 是正電荷與負電荷之間的吸引力，容易被水合破
壞。
b.氫鍵 (hydrogen bond) 是分子中的氫原子，因其 陰電性 太弱，原子核裸露
出來，而帶有正電荷，與帶負電荷的氧原子 (或氮原子) 之間，所生成的引
力。
c.疏水性引力 (hydrophobic bond)： 非極性分子具疏水性，兩個疏水性分子，
因受環境極性水環境的排斥，其分子間會生成 非極性-非極性 的疏水性引
力；水溶液中的巨分子，其疏水性引力多發生在分子內部。
d.凡得瓦爾力 (van der Waals bond)： 非極性或極性很弱的分子表面，其原子
受到鄰近分子上面原子的影響 (吸引或排斥)，會產生局部且短暫的偶極，
因而有微弱的引力，是為凡得瓦爾力。 兩個原子的距離要適中，以求得最
大的凡得瓦爾力，稱為該原子的 凡得瓦爾半徑。 兩分子之間因 構形互補
所生成的專一性吸引力，主要是由許多凡得瓦爾力所共同構成的。
胺基酸
胺基酸是構成蛋白質的基本單位，蛋白質是生物体內最重要的活性分子，其中擔
任催化生理代謝反應的酵素，更是近代生物化學的研究中心。 二十種性質各異
的胺基酸，連接組成多樣的蛋白質，且賦予蛋白質特定的分子構形，使蛋白質分
子能夠具有生化活性。Zwitterion (雙性離子)用以描述其電荷特性。
1.胺基酸基本構造：
胺基酸種類很多，但有共同的基本構造；先畫一個十字，如下述方法在四端
加上四個化學基團即可。在這十字的中央填上一個碳原子 (叫做 α 碳)，在周圍
的四個位置分別填上一個氫原子、胺基、羧基及一個 R 基團，基本的胺基酸構
造即完成。
R 基團可以由最簡單的 H 開始填入，就是最簡單的 glycine；再來是 CH3
就是 alanine；如此越來越大，並加入其它種類的原子 (如氧或硫)，或額外的胺
基或羧基，就可以組成多采多姿的二十種胺基酸。請注意 a 碳是不對稱的，因
為它周圍的四個原子或基團都不相同；只有當 R 基團為氫原子時，是對稱的 a
碳 (因為接有兩個一樣的氫原子)；也就是說只有 glycine 是對稱的胺基酸。 因
此，除了 glycine 外，其它胺基酸都有其立體異構物，兩立體異構物間的化學式
完全一樣，但互相成為鏡像，胺基酸的立體異構物以 L- 及 D-form 來表示之；
地球上的生物大都採用 L-from 胺基酸。
a.分子構造的中心為一碳原子，稱為 a 碳 (a carbon)。
b.接在 a 碳上，有一個 胺基 及一個 酸基 (故名胺基酸)。
c.另有一氫原子及一基團 (R) 接在 a 碳上 (碳為 sp3 軌道)。
d.α 碳接了四個不同的基團，為 不對稱碳，有 光學異構物 (D/L)。 通常細胞
的代謝只使用 L 型胺基酸，但有些細菌細胞壁或抗生素上，有 D 型胺基
酸。
e.隨 R 基團的不同，各胺基酸的性質互有差異，組成二十種胺基酸。
2 胺基酸分類：
胺基酸由其 R 基團的化學構造不同，可分為數大類。
a.胺基酸的基團形形色色，有大有小、有直鏈有環狀、有正有負也有不帶電。
表 1 中列出蛋白質中所用的二十種胺基酸，是以 R 基團的化學構造來分
組。
b.R 基團也可以其極性大小來分類，代表它們親水性的強弱；可把胺基酸分為
極性 及 非極性 兩大類，極性者又分為 酸性、中性、鹼性 三類。
c.胺基酸本身的性質，以及所組成蛋白質分子的功能與性質，均決定於 R 基
團的本質。
 表 1 二十種胺基酸的分類及性質：

分 類 名 稱 縮寫 R= 極性 (1)

唯一對稱胺基酸 甘胺酸 Glycine Gly G -H (構造最簡單) P/N

丙胺酸 Alanine Ala A -CH3 N
纈胺酸* Valine Val V -C(C)-C N*
含飽和碳氫基團
白胺酸* Leucine Leu L -C-C(C)-C N*
異白胺酸* Isoleucine Ile I -C(C)-C-C N*
苯丙胺酸* Phenylalanine Phe F -C-[C6H5] N*
酪胺酸 Tyrosine Tyr Y -C-[C6H4]-OH P
含芳香基團
色胺酸* Tryptophan Trp W -C-[indole] N*
組胺酸* Histidine His H -C-[imidazole] P*
天冬胺酸 Aspartic acid Asp D -C-COOH P
天冬醯胺
含額外酸基 [Asparagine] Asn N -C-CONH2 P
酸

[及其醯胺] 麩胺酸 Glutamic acid Glu E -C-C-COOH P

麩醯胺酸 [Glutamine] Gln Q -C-C-CONH2 P
離胺酸* Lysine Lys K -C-C-C-C-NH2 P*
含額外胺基
精胺酸* Arginine Arg R -C-C-C-[guanidine] P*
絲胺酸 Serine Ser S -C-OH P
穌胺酸* Threonine Thr T -C(OH)-C P*
含有醇基
OH- 脯 胺
Hydroxy Pro P
酸

甲硫胺酸* Methionine Met M -C-C-S-C N*

胱胺酸 Cysteine Cys C -C-SH P
含有硫
雙胱胺酸 Cys-Cys
Cystine -C-S-S-C 雙硫鍵
(3) (2)

環狀的亞胺酸 脯胺酸 (3) Proline Pro P (imino acid) N

(1) 打有 * 者是必需胺基酸，須由外界攝取；極性大小 N, non-polar; P, polar。

(2) 兩分子胱胺酸 (Cys) 以 雙硫鍵 連成二元体 (Cys-Cys)。

(3) 這兩個胺基酸對蛋白質的立體構造有很大的影響。
3.胜 肽：
胜肽是較短的蛋白質，許多胜肽有重要的生物功能或活性。
a.胜鍵 (peptide bond) 是由一個胺基酸的酸基，與次一胺基酸的胺基，行 脫水縮
合反應 而成的 C-N 鍵，具有雙鍵的性質，與相鄰總共六個原子在同一平面
上，因此 C-N 鍵不能自由轉動；胜鍵是構成蛋白質架構的基本單位，非常重
要，請注意研究其立體構成。 Peptide bond 的生成 (是一種脫水反應);
Peptide bond 的性質 (兩個 p 軌道 電子的共振)
b.兩個胺基酸以胜鍵連成的二元体，稱之為 雙胜 (dipeptide)，三個胺基酸則以
兩個胜鍵連成 三胜 (tripeptide)，許多胺基酸連成 多胜 (polypeptide)；再大的
胜肽即為蛋白質。
c.某些胺基酸或胜肽具有較特殊的生理活性，如 味素 (Glu)、腦啡 以及部份 荷
爾蒙 等。具有生理功能的 peptides (許多短鏈 peptides 即具有生理活性)

4.胺基酸的離子性質：
胺基酸多以離子狀態存在，且經常同時帶有正電及負電基團。

4.1 解離度 (pKa)：

質子是化學層次最小的粒子，很容易由一極性基團解離出來，在水溶液中無
所不在；其解離難易可以解離度 (pKa) 表示之，水的 pKa 在 6-7 之間。
a.質子搶奪：
氫原子若與陰電性大的原子 (如酸基 -COOH 中的氧原子) 共價，則其電子
易遭搶奪而使質子裸露 (-COO-H+)，進而解離成 H+。 質子又易受帶有高
電 子 密 度 的 基 團 ( 如 -NH2+) 所 吸 引 ， 使 後 者 成 為 一 帶 有 正 電 的 基 團
(-NH3+)。弱酸才可作為緩衝液分子
b.Ampholyte：
胺基酸的酸基易解離出質子 (成為帶負電基團 -COO-)，而其胺基又會接受
一 質 子 ( 成 為 -NH3+) 。 如 此 一 分 子 同 時 帶 有 正 電 與 負 電 者 ， 稱 為
ampholyte。質子可以吸附或脫離一基團 (因此溶液中質子濃度可以改變)
c.質子解離：
解離程度決定於該水溶液的 pH 與分子上解離基團 pKa 的高低。 pKa 值
的大小，顯示一個官能基容不容易放出 H+，越小的越容易放出。 圖 3 列
出各種胺基酸的解離基團及其 pKa，請注意各種基團在不同的 pH 下解
離。當環境的 pH 等於某基團的 pKa 時，該基團恰有一半數目的分子解
離 (pH = pKa + log ([A-]/[HA])。

4.2 等電點 (pI)：

等電點是所有細胞分子帶電性質的重要指標。
a.官能基可能帶有電荷：
胺基酸 a 碳上的胺基及酸基各有一帶電基團，故有二 pKa，分別界定胺基及
酸基的解離 pH。此二 pKa 平均值即為該胺基酸的 pI (等電點)，即 (pKa1 +
pKa2) ÷ 2 = pI。
b.等電點：
若環境的 pH 等於某胺基酸的 pI，則此胺基酸的 淨電荷 為零；因為在此
pH 下，剛好有一正電基團及一負電基團。 Ampholytes 中淨電荷為零者，
其正、負電基團數目相等，特稱為 Zwitterion。
c.環境控制胺基酸的淨電荷：
胺基酸的淨電荷是正是負，受環境的 pH 所控制；環境 pH > pI 帶負電，
反之則帶正電。請注意環境的 pH 離該分子的 pI 越遠，則其所帶之正或負
淨電荷越大。
d.有三個官能基的胺基酸：
某些胺基酸的 R 基團，有額外的帶電基團 (例如 Lys 另有一胺基)，則可
有三個 pKa；即每個可解離出 H+ 或可吸收 H+ 的官能基，都有一個 pKa。
這三個 pKa 中，有兩個 pKa 的胺基酸帶一個淨正電或淨負電，則這兩個
pKa 值的平均即為其 pI。
e.如何計算多肽的淨電荷：
多肽在某 pH 下的淨電荷，是所組成的胺基酸各基團所帶正、負電荷的總
和。例如一條十胜所含的十個胺基酸 (AELKVGRRDV) 中，若有五個胺基
酸為非極性，三個帶正電基團，兩個帶負電，則此十胜在中性 pH 下的淨電
荷為一個正電荷。
蛋白質
蛋白質有各種催化及生理機能，是細胞的主要工作機器，其功能乃源自蛋白質分
子所具有的 特定構形 及 催化活性；此種構形的形成，又因於組成蛋白質的胺
基酸排列次序。 各種長短的蛋白質有不同的胺基酸組成與排列，造就了多樣而
多功的蛋白質繽紛世界。

A.蛋白質構造：
像其它巨分子一樣，蛋白質鏈也是由小分子單位 (胺基酸) 一個一個連接成的。

1.一級構造：
探究蛋白質構造可由胺基酸序列開始，循序依其複雜度分成四個層次。

a.蛋白質的骨架：
蛋白質的長條胺基酸序列，是為其 一級構造 (primary structure)。 此一級構造
的 一 端 為 N- 端 (-NH2) ， 另 一 端 為 C- 端 (-COOH) ， 而 以
H2N-C-C-[N-C-C]x-N-C-COOH 為骨架，其中黑體字 C 代表各胺基酸單位的
α 碳。
一級構造︰蛋白質的骨架 (N-C-C-N-C-C-N-C-C-N-)
b.一級構造信息是由 DNA 所決定：
一級構造是蛋白質最終 構形 的根本，各級構造的訊息都決定於胺基酸的序
列；而胺基酸序列是根據 DNA 的核苷酸序列轉譯而來，故最終的信息是存
留於細胞核的核酸中。
c.蛋白質序列可由核酸序列得知：
探討一級構造主要在分析該蛋白質之 胺基酸序列，近來胺基酸序列多由該蛋
白質的 cDNA 經分子群殖 (cloning) 篩選出，再經核酸定序後轉譯成胺基酸
而來。
d.有三個官能基的胺基酸：
除了構造之外，一段固定的胺基酸序列可能有某種特定的生理功能，稱之為
signal peptide (信息序列)，同一 signal 可以在許多不同的蛋白質分子上重複出
現。 例如蛋白質在 C-端若有 Lys-Asp-Glu-Leu (KDEL) 的序列，則會被回收
到內質網去。

2.二級構造：
蛋白質長鏈捲繞成堅固而規則的二級構造，是其構形的基礎單位。

（一）胜鍵的雙鍵性質形成 胜鍵平面；又因兩 α 碳上的 R 基團與前後相鄰基

 團的引力或斥力，使得兩相鄰胜鍵平面間的轉動，限制在一定角度範圍，
而造成規律的兩種主要構形：α 螺旋 (α helix)、 b 長帶 (b sheet)，是為 二
級構造 (secondary structure)，它們的主要構成力量都是 氫鍵。 氫鍵在細
胞內的角色不但重要，而且分佈非常廣泛。
a.α 螺旋：
每 3.6 個胺基酸捲繞一圈，成為右手旋的螺旋構造，遇 Pro 則中止；由
相鄰兩胜鍵平面所夾的角度，可以預測 α 螺旋或其它二級構造的生成
(Ramachandran Plot)。 分子內氫鍵可在螺旋骨架間加上支架，更使得 α 螺
旋成為圓筒狀，有堅固的構形，也是三級構造的組成單位之一。 肌紅蛋白
由八段長短不等的 α 螺旋所組成。
b.β 長帶：
像彩帶般的構形，多由數條彩帶組合而成，相鄰彩帶之間以 氫鍵 接合，編
成一片堅固的盾形平面。 依相鄰彩帶的 N→C 方向關係，可分為 同向
(parallel) 及 逆向 (antiparallel) 兩大類。β 長帶多由 R 基團較小的胺基酸
(如 Ala, Gly, Ser) 組成。
二級構造︰βsheet, turns (也是由氫鍵構成)
c.其它螺旋構造：
除了 α 螺旋外，蛋白質也可以其它方式摺疊成螺旋構造，但每一單位螺旋的
胺基酸數目不同。 當連續有數個 Pro (如 PVPAPIPP) 時，會捲成稱為
polyproline 的螺旋，每三個胺基酸轉一圈，橫切面是一個正三角形。

（二）連結性二級構造：
a.Turn 轉折：
連接 α 螺旋或 β 長帶時，胜肽鏈需做劇烈的轉折，以接近 180 度的方式摺
返，這些轉折點稱為 turns；其中 β turn 由四個胺基酸組成 (多含有 Gly)；
γ turn 則由三個胺基組成，且由一個 Pro 造成主要轉折。 Turns 多分佈在
蛋白質的表面，也很容易誘生抗體。
b.不規則形：
除上述構造外，尚有構形不規則的連結片段，稱為 不規則形；而在蛋白質
分子兩個端點附近的胜肽，活動性較大，形狀經常變化，則為 任意形
(random coil)。
二級構造︰β sheet, turns (也是由氫鍵構成)

3.三級構造：
大部分蛋白質的三級構造捲繞成 球狀 (globular)，已是有特定構形的活性分
子。
（一）三級構造的組成力量：
a.二級鍵組合三級構造：
 分子內各部分的二級構造再相互組合，構成完整球形的 三級構造 (tertiary
structure)；其構成的作用力有 離子鍵、氫鍵、疏水鍵、金屬離子 等作用
力；其中疏水鍵對水溶性蛋白質三級構造之穩定性，貢獻最大。
b.疏水鍵穩定蛋白質核心：
水溶性蛋白質的核心緊密，多由疏水性胺基酸組成。 由於疏水性胺基酸為外
界水環境所排斥，可以穩定地包埋在分子內部，維持蛋白質的完整三級構
造。

c.雙硫鍵加強構造：
蛋白質分子內兩個 Cys 上的 -SH 可經氧化而成 雙硫鍵 (-S-S-)，雙硫鍵可加
強蛋白質的立體構造。 在細胞中，雙硫鍵的形成可能需要靠酵素的催化。

（二）三級構造的立體構成：
a.Domain 的形成：
某些二級構造經常會聚在一起，例如 aaaa, bab 或 a8b8 筒狀構造等，都可自
形成一小區域，稱為 domain (功能區塊)，若干 domains 再組成一完整蛋
白質的三級構造。 小的蛋白質通常只有一個 domain，較大蛋白質則含有
兩個以上的 domains。 經常重複出現的 domain，可能有特定功用，可視
為一種二級構造的再現單位 (motif)。
b.蛋白質構形的資訊儲藏在胺基酸序列中：
建構蛋白質最終立體構形的藍圖，其資訊是貯藏在一級構造的胺基酸序列中；
因此某些蛋白質 (如 RNase) 在 變性 後，仍然可以回復原來的立體構形
(原態 native)，稱為 復性。
例子：RNase, myoglobin。
c.Chaperone 幫助摺疊正確構形：
並非所有的變性蛋白質都可如 RNase 般復性回原態，細胞內有一類巨大分子
稱為 chaperone，可以幫助蛋白質正確地摺疊成原態分子。
d.以胺基酸序列只能正確預測二級構造：
若已知某蛋白質的胺基酸序列，則可以電腦運算預知某段是 a 螺旋、b 長帶
或是轉折等二級構造，相當準確。 但更複雜的三級立體構造，目前則較不容
易預測準確。
（三）三級構造的修飾：
a.蛋白質再經修飾：
很多蛋白質的三級構造，即為獨立而具有活性的分子； 但有些則要再加上 輔
酶、輔因子 (如 金屬離子) 或 輔基 (prosthetic group, 如 肌紅蛋白 中的
heme) ； 有 些 則 要 再 修 飾 以 糖 分 子 成 為 醣 蛋 白 (glycoprotein) ； 脂 蛋 白
(lipoprotein) 要連接脂質；更有的要再與其它相同或不同的蛋白質分子結合，
形成四級構造 (如 血紅蛋白)。
b.有些蛋白質以裂解產生活性：
有些蛋白質的胺基酸鏈要先經過切斷或刪除某段胜肽後，才能有活性 (如 胰
島素)。胰島素是由一條基因轉譯出來的，切成三段後，其中兩段以雙硫鍵結
合成具活性的三級構造，並非四級構造；這是蛋白質活性的調控方式之一。

4.四級構造：
蛋白質再聚合成四級構造，可調節控制其功能，或組成巨大構造分子。
a.次體組成四級構造：
若數個相同或不同的三級構造分子，再結合成一較大的複合體，才能進行完整
的活性功能，則稱為 四級構造 (quaternary structure)。 構成四級構造的每一
單位分子，稱為 次體 (subunit)；通常各次體之間無共價結合，而以二級鍵為
主要結合力量。每一條次體蛋白質，都是由一個完整基因所轉譯出來的。
b.四級構造之目的：
若四級構造的任一次體與受質結合之後，會誘導增強其它次體與受質之結合能
力，而加速反應，則稱為 正協同作用 (positive cooperativity)，反之則為 負協
同作用。 四級構造在 調控 蛋白質的活性上非常重要，許多酵素是重要的例
子；但典型具四級構造，且有複雜調控機制的血紅蛋白並非酵素。
c.構成性四級構造：
許多病毒的蛋白質外殼，具有規律而巨大的四級構造組成。

B.蛋白質性質：
蛋白質要有正確的分子 構形，才能有效執行其生理功能；構形 或許是分子演化
的基本驅策力，因為即使胺基酸序列不十分相似，同功能的蛋白質也可能
有相同的 構形 (各種生物的血紅蛋白即為一例)。

1.變性及復性：
某些條件會破壞蛋白質分子的各級構造，稱之為 變性 (denatuaration)，例如 加
熱、pH 太 高或太低、尿素、界面活性劑、劇烈震盪等。變性的蛋白質大多會失
去活性，當變性條件除去後，有些蛋白質會回復原來構形，並具原有活性，稱之
為 復性。
～原態與變性 (蛋白質的最主要性質)
～SDS 破壞三級構造的疏水性核心 (一頭進入蛋白質內部 一頭在外親水)
～Mercaptoethanol 也是抗氧化劑 (小心使用 過多反而有害酵素)
2.蛋白質構形是活動的：
蛋白質分子上的各部份結構並非固定不動，而是有相當的 彈性與運動。 尤其
domain 與 domain 之間，或者酵素催化區的開閤，都有相當大的活動幅度。
這種活動會隨著溫度升高而上升，對蛋白質或酵素的活性及其調控有很大
的影響。
3.蛋白質的專一性結合：
專一性在酵素的催化及細胞生理功能上，扮演重要角色。蛋白質與蛋白質之間，
或與其它分子 (例如 核酸或者細胞膜)，經常有專一性的結合，其構成力量
如下：
a. 構形互補 (conformational match)：
兩分子間的結合表面，其形狀互補，像拼圖積木。
b. 二級鍵吸引力 (interaction forces)：
兩分子之結合面上，對應胺基酸間的吸引力量，由二級鍵構成。 圖 1 是一
假想圖例，說明某酵素與其抑制因子間，如何進行專一性的結合。

C.蛋白質研究技術：
蛋白質研究通常要先純化得均質蛋白質，然後檢定其分子量、次體組成及等電
點，最終則要定出蛋白質之胺基酸序列，或其立體三次元分子構造。

1. 蛋白質純化技術：
利用蛋白質分子量不同、表面帶電性或極性區域大小等 性質差異，可分離純化
之。通常以能夠純化出大量均質蛋白質為目標，但最近的純化觀念已稍有
改變，以二次元電泳或加上轉印，直接挖出單一點的蛋白質，可馬上進行
分析 (proteomics 的主要實驗操作)。
a. 硫酸銨分劃法：
在蛋白質的水溶液中加入硫酸銨鹽類，會使蛋白質因疏水性區域相吸引而聚
集沉澱出來，稱為 鹽析 (salting out)；鹽析大略地純化出蛋白質，並可以除
去核酸、醣類或脂質等物質。
b. 膠體過濾法 (gel filtration)：
依蛋白質分子量的大小不同，先後分離出來。
c. 離子交換法 (ion exchange)：
各種蛋白質的帶電性強弱不同，與離子交換介質間吸引力的大小會有差異，
可以進行分離。蛋白質的帶電性，會因環境的 pH 不同而有改變。
d. 親和層析法 (affinity chromatography)：
利用分子間專一的親和性來吸引純化某蛋白質，最為直接；但並非所有蛋白
質都能夠找到專一性的吸著劑。

2.蛋白質性質與構造檢定：
a.蛋白質定量法：
染料 Coomassie Blue 與蛋白質結合後，會由褐色變為藍色；由反應前後藍色
吸光度的改變，與已知蛋白質的標準曲線比較，即可推知樣本中蛋白質的
濃度；現在一般多使用
Bradford's method。
b.分子量測定法：
可利用 膠體過濾法、超高速離心法 (ultracentrifugation)、或 膠體電泳法 (gel
electrophoresis) 來測定蛋白質的原態分子量；SDS 膠體電泳 則可測定次體
分子量。 電泳 同時可以檢定蛋白質的純度如何，是解析力極佳的分析工具。
c. 等電點 (pI)：
等電焦集法 (isoelectric focusing) 極類似膠體電泳，但可測得蛋白質的等電
點。 等電點是蛋白質帶電性質的重要指標，當環境的 pH 等於其等電點時，
此蛋白質的淨電荷為零；大於其等電點時，淨電荷為負，反之則為正電荷。
d. 胺基酸組成：
蛋白質以鹽酸水解成游離胺基酸，再以分析各種胺基酸之含量。
e. 蛋白質立體構造：
以 X 光繞射法 分析蛋白質結晶，可計算出其分子的細微立體構造；近來也
流行應用 核磁共振法 (NMR) 測定水溶液中蛋白質的立體構造。
f. 質譜分析：
目前的質譜儀分析技術，已經能夠處理分子量較大的蛋白質，則可以定出蛋
白質的精確分子量；甚至可以檢查該蛋白質所產生的各個片段，推出其胺基
酸序列。

3.蛋白質序列決定法：
胺基酸序列 是一個蛋白質的最根本資料，只要定出其胺基酸序列，就可以推出
相當多的生化性質。

（一）傳統胺基酸定序法： 傳統的胺基酸定序方法，是直接檢定胜肽鏈上的胺
基酸種類。
a. Edman degradation：
許多化學反應 (如 Edman degradation) 可由蛋白質的 N-端開始，依序一次切
下一個胺基酸，再檢定每輪切下的胺基酸為何，即可推得此蛋白質的胺基
酸次序。現在都用自動定序儀器，最多可定到 50 個胺基酸。
b. 蛋白質通常要先切成片段：
若蛋白質太長，則無法有效定序後面的胺基酸序列；要先用蛋白質水解酶把
目標蛋白質切成小段，各小段分別定序，然後再組合成長鏈。
c. 以重疊片段排序：
為了排列上述各小段胜肽的先後次序，要使用兩種不同的蛋白質水解酶，得
到兩套不同長短的胜肽，分別定序後，比較各片段重疊部分，即可判斷先
後次序。

（二）cDNA 定序：
以基因操作方法，選殖出目標酵素的 cDNA，並將其核酸序列定出，則可推出所
 對應的胺基酸序列；目前大都採用此種方法。

（三）以質譜儀定序：
質譜儀是利用分子的質量大小來檢定樣本，因此可以精確測出某分子的質量。若
把蛋白質在質譜儀中撞擊，產生一群具有各種不同長短的片段，每一片段
都剛好少一個胺基酸，然後用質譜儀一一測出這些片段的分子量，由所得
各種片段分子量的差別，就可推出相差胺基酸的種類，乃至整段胺基酸的
序列。
Oxygen Delivery and Storage

The protein hemoglobin serves as the oxygen carrier in the blood.

Hemoglobin provides a means of getting oxygen to metabolizing tissues, but cells also
need a means to bind and store oxygen released from the hemoglobin. This function is
carried out by the intracellular protein myoglobin, a structural relative of hemoglobin.
The hemoglobin in the red blood cells and myoglobin inside tissues such as muscle
cells have to act in tandem for effective oxygen transport.

The following graph illustrates the different oxygen affinities of myoglobin and
hemoglobin at different concentrations of oxygen (given as partial pressure of O2)

Note how the hemoglobin dissociation curve is S-shaped, or sigmoidal, in character.

Myoglobin is found intracellularly in body tissues.
Myoglobin (Mb) must effectively bind any oxygen released from hemoglobin (Hb).
Myoglobin therefore needs to have a higher oxygen affinity than hemoglobin..

Myoglobin is a monomeric heme protein found mainly in muscle tissue where it

serves as an intracellular storage site for oxygen.

The tertiary structure of myoglobin is that of a typical water soluble globular protein.
Its secondary structure is unusual in that it contains a very high proportion (75%) of
-helical secondary structure. A myoglobin polypeptide is comprised of 8 separate
right handed -helices, designated A through H, that are connected by short non
helical regions.

Each myoglobin molecule contains one heme prosthetic group inserted into a
hydrophobic cleft in the protein. Each heme residue contains one central coordinately
bound iron atom that is normally in the Fe2+.
The oxygen carried by hemeproteins is bound directly to the ferrous iron atom of the
heme prosthetic group.
Carbon monoxide also binds coordinately to heme iron atoms in a manner similar to
that of oxygen, but the binding of carbon monoxide to heme is much stronger than
that of oxygen. The preferential binding of carbon monoxide to heme iron is largely
responsible for the asphyxiation that results from carbon monoxide poisoning.

Hemoglobin is an [ (2): (2)] tetrameric hemeprotein found in erythrocytes where it

is responsible for binding oxygen in the lung and transporting the bound oxygen
throughout the body. Each subunit of a hemoglobin tetramer has a heme prosthetic
group identical to that described for myoglobin.

The quaternary structure of hemoglobin leads to physiologically important allosteric

interactions between the subunits, a property lacking in monomeric myoglobin which
is otherwise very similar to the -subunit of hemoglobin.
The curve of oxygen binding to hemoglobin is sigmoidal typical of allosteric proteins
in which the substrate, in this case oxygen, is a positive homotropic effector.

cooperativity
When oxygen binds to the first subunit of deoxyhemoglobin it increases the affinity of
the remaining subunits for oxygen. As additional oxygen is bound to the second and
third subunits oxygen binding is further, incrementally, strengthened, so that at the
oxygen tension in lung alveoli, hemoglobin is fully saturated with oxygen.

As oxyhemoglobin circulates to deoxygenated tissue, oxygen is incrementally

unloaded and the affinity of hemoglobin for oxygen is reduced.

The oxygen binding curve for myoglobin is hyperbolic in character indicating the
absence of allosteric interactions in this process.
The allosteric oxygen binding properties of hemoglobin arise directly from the
interaction of oxygen with the iron atom of the heme prosthetic groups and the
resultant effects of these interactions on the quaternary structure of the protein.

When oxygen binds to an iron atom of deoxyhemoglobin it pulls the iron atom into
the plane of the heme. Since the iron is also bound to histidine F8, this residue is also
pulled toward the plane of the heme ring. The conformational change at histidine F8 is
transmitted throughout the peptide backbone resulting in a significant change in
tertiary structure of the entire subunit.

Conformational changes at the subunit surface lead to a new set of binding

interactions between adjacent subunits. The latter changes include disruption of salt
bridges and formation of new hydrogen bonds and new hydrophobic interactions, all
of which contribute to the new quaternary structure.
The tertiary configuration of low affinity, deoxygenated hemoglobin (Hb) is known as
the taut (T) state. Conversely, the quaternary structure of the fully oxygenated high
affinity form of hemoglobin (HbO2) is known as the relaxed (R) state.

These effects of hydrogen ion (proton) concentration are responsible for the well
known Bohr effect in which increases in hydrogen ion concentration decrease the
amount of oxygen bound by hemoglobin at any oxygen concentration (partial
pressure).
Tissue CO2 is also carried to the lungs as the dissolved gas and as bicarbonate formed
spontaneously and by the enzyme carbonic anhydrase which converts CO2 and H2O to
carbonic acid. The carbonic acid thus formed spontaneously ionizes to proton and
bicarbonate ion:
CO2 + H2O --------> H2CO3 ------> H+ + HCO3-

The compound 2,3-bisphosphoglycerate (2,3-BPG), derived from the glycolytic

intermediate 1,3-bisphosphoglycerate, is a potent allosteric effector on the oxygen
binding properties of hemoglobin.

2,3-BPG can occupy this cavity stabilizing the T state. Conversely, when 2,3-BPG is
not available, or not bound in the central cavity, Hb can be converted to HbO2 more
readily. Thus, like increased hydrogen ion concentration, increased 2,3-BPG
concentration favors conversion of R form Hb to T form Hb and decreases the amount
of oxygen bound by Hb at any oxygen concentration.

HbF (the fetal form of hemoglobin) binds 2,3-BPG much less avidly than HbA (the
adult form of hemoglobin) with the result that HbF in fetuses of pregnant women
binds oxygen with greater affinity than the mothers HbA, thus giving the fetus
preferential access to oxygen carried by the mothers circulatory system.
Sickle-cell anemia: anemia，血紅蛋白結構中的β鏈有一個胺基酸由正常的穀胺酸
殘基（glutamate）被取代成纈胺酸殘基（valine），根據血紅素(hemoglobin)
的晶體結構，這個突變位於血紅素分子的表面，纈胺酸殘基的疏水性支鏈，正好
可以插入另一個血紅素分子表面上疏水性的凹洞。這種血紅素分子間的聚合會導
致纖維化現象，而造成紅血球變形成鐮刀狀，無法正常地輸氧。

地中海貧血，又稱作海洋性貧血,因其原文 Thalassemia 在希臘文裏就是海洋的

意思,它是一種遺傳性的貧血,因首見於地中海沿岸種族,故名之謂地中海貧血.
好發地區包括地中海,中東,印度洋及南中國海沿岸國家,與人種有關,中國則見
於南部各省份,如雲南.廣東.廣西.福建及包括台灣.地中海貧血目前是世界上最
多見的遺傳性疾病,遺傳帶因的人口達到兩億人以上.當α-chain 或β-chain 基因
點發生遺傳缺憾時,就會無法合成正常的成人 Hb,自然形成貧血.若α-chain 基因
點有缺憾,稱作α-地中海貧血,反之β-chain 基因點有缺憾,稱作β-地中海貧血。
酵素
1. 酵素概述
Enzyme 一字源自希臘文，原意為 "in yeast"； 描述在酵母菌中，含有某種神奇
的催化活力，可以把糖轉變為酒精，故名為酵素。 Sumner 在 1926 年首先結晶
出 尿素酶 (urease)，並證實酵素為一種蛋白質。 一般而言，酵素具有下列特性：
a. 酵素可催化生化反應，增加其 反應速率，是最有效率的催化劑。
b. 酵素種類非常多，每一種都能催化所賦與的 專一性 反應，其它的酵素不易
干擾；不過，可能會有酵素間的協同或抑制作用。
c. 酵素的催化反應是 可調節 的，反應可受許多因子影響而加快或減緩。
d. 通常酵素為 蛋白質，但部份 RNA 也具專一性的催化能力 (ribozyme)。
生物體藉著種種酵素的催化作用與調節，才能有效地完成他所需要的許多生理活
動。 若細胞內的酵素活動受到抑制或干擾，整個生物體就可能出現異狀。

2.酵素的命名：
酵素的命名，有一定規則可循。

a.早期命名：
最初酵素命名並無法定規則，但都附有 -in 或 -zyme 等字尾，例如 trypsin,
renin 及 lysozyme 等；後來漸以該酵素催化的反應加上 -ase 字尾為名，再冠
上此反應的反應物，如 histidine decarboxylase (反應物 + 反應-ase)。
b.系統命名法：
1965 年命名系統化，把所有酵素依催化反應分成六大類，以四組數字名之
(IUBMB 系統)；例如 histidine carboxylase 為 EC 4.1.1.22：
Main Class： 4 Lyases 分裂 C-C, C-O, C-N 鍵
Subclass： 4.1 C-C lyase 分裂 C-C 鍵
Sub-subclass： 4.1.1 Carboxylase 分裂 C-COO 鍵
序列號碼： 22 第 22 個 4.1.1 分裂組胺酸的 C-COO 鍵
c.IUBMB 系統所分的六個 Main Classes：
電子或質子
EC1 Oxidoreductase 氧化還原酶
轉移
官能基團的
EC2 Transferase 轉移酶
轉移
加水或脫水
EC3 Hydrolase 水解酶
分子
共價鍵生成
EC4 Lyase 裂解酶
或裂解
 同一分子內
EC5 Isomerase 異構酶
基團之轉移
消 耗 ATP
EC6 Ligase 連接酶 生成分子間
新鍵

3.酵素的構成：
酵素主要由蛋白質所構成，不過許多酵素還需加上其它物質；有些 RNA 也具有
催化的能力，在分子演化上可能是最早出現在地球上的巨分子。
A.全酶：
全酶是具有完整分子構造及催化能力的酵素。
a.全酶的組成：
一般酵素由蛋白質構成，但某些酵素為 醣蛋白 或 脂蛋白，有些要加上 輔助
因子 (cofactor, coenzyme)，才成為功能完全的酵素 (全酶 holoenzyme)；若全酶
失去了輔助因子，剩下的部份稱為 apoenzyme：

Holoenzyme ＝ Apoenzyme ＋ Cofactor / Coenzyme

b.各種形式的酵素組成構造：
全酶分子可能只含一條多肽，也可能含數條多肽，並以 雙硫鍵 連接在一起 (如
chymotrypsin)；有的可由數個相同或不同的 次體 (subunit) 組成。 肝糖磷解酶
為同質二元體 (dimer)；而 血紅蛋白 (hemoglobin) 是 a2b2 的四元體形式，但
並非酵素。 多元體蛋白質可能具有 異位調節 功能 (allosteric effect)，即任何
一個次體改變，會影響其它各個次體的活性。

B.輔酶：
一些非蛋白質的小分子會加入酵素構造中，以幫助催化反應進行。因為二十種胺
基酸的官能基中，具有強荷電性者不到五個，而酵素活性區經常需要較強的官能
基來引發催化反應，部份酵素因此納入蛋白質以外的輔助因子參與其構造，作為
催化的重要反應基團。

（一）輔助因子：
包含金屬離子以及小分子的有機物質 (輔酶)。
a.金屬離子：
如 Zn2+, Mg2+, Mn2+, Fe2+, Cu2+, K+，以離子鍵結合在 His, Cys, Glu 等胺基
酸；細胞多使用較輕的金屬，重金屬多有害處。
～金屬離子可維持構形 (可結合胺基酸 Glu, Asp, His, Cys)
b.有機小分子：
 分子構造稍複雜而多樣，又稱為 輔酶 (coenzyme)，哺乳類多由維生素代謝而
來，無法自行合成；如 維生素 B 群、葉酸 (folic acid)、菸鹼酸 (niacin)。

（二）輔酶的作用：
輔酶的構造與其功能極為重要，請注意每一種輔酶的特定作用機制。
a.改變酵素構形：
加入酵素分子，誘使改變其立體構形，而使得酵素與基質的結合更有利於反應。
b.協助催化反應：
輔酶可作為另一基質來參與反應，但反應後輔酶構造不變。 通常輔酶作為某特
定基團的轉移，可供給或接受基團 (如 -CH3, -CO2, -NH2) 或者電子，這類輔
酶最是常見。
c.直接提供反應基團：
提 供 一 個 強 力 的 反 應 基 團 ， 吸 引 基 質 快 速 參 加 反 應 ； 例 如 維 生 素 B1
(thiamine)，有許多維生素都是輔酶。

（三）輔助因子範例：
a. Dehydrogenases：
各 種 去 氫 酶 (dehydrogenase) 以輔酶 NAD+/NADH 轉運 hydride ；要研究
alcohol dehydrogenase 及 glyceraldehyde-3-P dehydrogenase 的作用模式，同時
請瞭解 NAD+/NADH 及 hydride (H-) 的構造。
～輔酶 NADH (催化去氫反應的重要輔酶)
～Hydride, H- (含有額外電子的氫原子) (擁有電子才有化學能)
b.Carboxypeptidase：
Carboxypeptidase 分子需要一個鋅離子維持分子構形 (induced fit)，同時也參與
催化反應，可以抓住基質胜肽，並活化水分子。
c.Glutarmate transaminase：
Glutamate transaminase 使用輔酶 pyridoxal phosphate 轉運胺基。
d.Catalase：
Catalase 分子上有一 Fe2+ 作為電子暫存區，可以把 H2O2 還原成水分子；而
血紅蛋白也有 Fe2+，因此可有類似的催化作用，但效率很低，因為其鐵離子
氧化成 Fe3+ 後無法很快變回。

（四）輔酶與 ribozyme：
輔酶的構造透露了遠古 RNA 分子的催化秘密：許多輔酶的構造中都有核苷酸參
與，可能是用來與遠古催化性 RNA 分子結合，以幫助 RNA 的催化反應；因為
ribozyme 雖然有分子構形，但缺乏催化所需的強烈官能基團，有如今日的蛋白
質酵素與其輔酶一般。
～Nucleotide 的構造 (磷酸 - 核糖 - 鹽基)
4.酵素動力學：
A.酵素催化反應：
酵素提供基質一個穩定的空間，有利於穩定其過渡狀態，並快速轉變成為生成物。

1. 酵素的催化反應
a.反應物 (A, B) 轉變成生成物 (A-B) 途中，有 過渡狀態 [A...B] 生成：
A + B → [A...B] → A-B
b.過渡狀態 (transition state) 的位能較高，其生成需要能量，稱為 活化能
(activation energy, Eact)；經由酵素的催化，可降低反應活化能，使反應速率加
快，但 不影響反應的平衡方向。
c.一些過渡狀態的類似物 (analog) 會卡住酵素活性區，但無法完成反應，即成為
抑制劑。 這種過渡狀態的類似物可做為抗原，免疫動物後所產生的抗體，可能
有類似酵素的催化作用，但催化速率較低，稱為 abzyme。
d.酵素降低活化能的機制有以下幾點，都是因為 活性區 的特殊立體構造：
(1) 酵素活性區專一性地與基質結合，提供最適的空間排列，以便穩定過渡狀
態。
(2) 活性區通常為一凹陷口袋，隔開外界的水環境，減低水分子的干擾。
(3) 活性區附近的某些胺基酸可提供 活性官能基 (通常帶有電荷) 直接參與
反應。
(4) 很多酵素含有 輔酶 或 輔因子，輔助反應。

B.酵素動力學：
酵素動力學的形成，是根基於『過渡狀態濃度恆定』的概念。 早在 1913 年，
Michaelis [發音 mi-ka-ei-lis] 及 Menten 就以 轉化酶 (invertase) 系統為研究對
象，發現有關酵素與基質反應的一些行為模式，他們提出：
a.Steady state 理論：
酵素催化時，基質先與酵素結合，生成過渡狀態，再轉變成產物； 而酵素與基
質的結合是 可逆的 (E + S → ES)； 而當反應達 穩定狀態 (steady state) 時，
其中的 [ES] 濃度不變 (因為 ES 生成量等於其消失量)。
～Invertase Reaction 轉化酶 (還原糖的測定法︰利用還原力)
～Steady State Theory 最重要基本假設 (有一 [ES] 濃度穩定狀態)
b.酵素行為的數學描述：
反應速率 (v) 與酵素或基質的關係，可以數學式表示； 在固定的酵素量下，
反應速率 v 與基質濃度 [S] 成雙曲線關係(但只有雙曲線一股)，可用公式表
之，即 Michaelis-Menten (M-M) 動力學公式。

1.Michaelis-Menten 公式的推演：
由四個基本設定開始，可一步一步推得 M-M 動力學公式。
 a.酵素 E 與基質 S 反應如下，各步驟反應速率由常數 k1, k2, k3 表示：

b.導 M-M 公式前的四個基本關係及假設：

(1) 因 [ES] 不變，故 ES 的消耗量等於生成量：
k2 [ES] + k3 [ES] = k1 [E][S] (I)

(2) 總酵素濃度 [Et] ＝ 單獨存在者 [Ef] + 酵素基質複合體

[ES] [Et] ＝ [Ef] + [ES] (II)

(3) 反應初速 (vo) 是由後半分解反應 (k3) 所決定：

vo = k3 [ES] (III)

(4) 最大反應速率 (Vmax) 是假設所有酵素均轉變成 [ES]，故上式可改寫為︰

Vmax = k3 [Et] (IV)
c.基於上述條件，可推 M-M 公式如下：

2. Michaelis-Menten 公式的意義：
M-M 公式可以求得 Vmax 及 Km，求得 Vmax 及 Km 有
 何意義？

a. M-M 公式是 雙曲線 公式。若固定酵素量，改變其基質量

[S]，則可得到不同的反應初速 vo，再以 [S] 為 x 軸，vo 為

y 軸作圖，可得到一股雙曲線，其漸近點為 Vmax。

b. 低濃度 [S] 時反應速率 vo 與 [S] 成正比，即 vo～[S] 1，

是為 一級反應 (first order reaction)； 當 [S] 增大，vo 接

近漸近線時，vo 的改變很小，不受 [S] 變化的影響，即 vo

～[S] 0，稱為 零級反應 (zero order)。

c. 若基質量 [S] 也固定，則 M-M 公式變為：

Vmax (常數 [S])
vo ＝ ────────── ～ Vmax (常數)
(常數 Km) + (常數 [S])
由 (IV) Vmax = k3 [Et]，故 vo～[Et]，即 反應速率 與 酵素量 成

正比。

d. 事實上 ES → E + P 的反應為可逆，但此逆反應可忽略，因 M-M 公式的測定，是反

應初期所測的反應初速 (vo)，此時生成物 [P] 的濃度很低，逆反應幾乎無從發生。

3. Vmax 及 Km 的測定與意義：
Vmax 及 Km 是每一個酵素極重要的性質指標，可以顯示其催化特性。

～Vmax 及 Km 測定法：
步驟相當單純，但隨著各種酵素活性測定方法的難易有別。動力學實驗的基
本資料，為在一系列 [S] 濃度下所測得的反應初速 (vo)，依法作圖即可求出
Vmax 及 Km。 有以下數種作圖求法：
a.直接作圖法：
是最基本的數據作圖。 以 [S] 為橫軸 vo 為縱軸，所得漸近線的最高處為
Vmax，Km 為 50% Vmax 時的 [S]。
b.Lineweaver-Burk 雙倒數作圖法：
是最常用的作圖方式；因直接作圖法只能以漸近估計求得 Vmax，若 x 及 y 軸
分別改為 1/[S] 及 1/vo，則可作出一條直線來，由 x 軸上的交點求出 1/Km，
由 y 軸交點求出 1/Vmax。
c. Eadie-Hofstee 作圖法：
雙倒數圖的直線，在接近 y 軸處，打點太密，求得直線稍有困難。 若分別以 vo
/[S] 及 vo 為 x, y 軸，亦可畫出直線，且各點的分佈較平均。

4. Km 的意義：
Km 是酵素與基質間親和力的指標，Km 越大親和力越小。
Km: 基質親和力 (前半段) (1/2 Vmax → Km = [S]； Km 大小)
a.當反應速率為 50% Vmax 時，vo = 1/2 Vmax，代入 M-M 公式，則得：
Vmax Vmax [S]
── = ────，整理得 Km ＝ [S] (只在 vo＝ 1/2 Vmax 條件下)
2 Km＋[S]
因此 Km 的意義表示，要達到一半最高催化速率時 [S] 所需濃度。
b.若酵素的 Km 越低，則表示它要接近 Vmax 所需的基質濃度越低。若某一酵
素有數種基質，各有不同的 Km，則 Km 越低的基質，表示它與酵素的親和力
越大，催化反應愈容易進行。 Km 與 [S] 一樣是濃度單位 (mM 或 mM)。
c.某酵素的 Km 值可看成在一般細胞內，該酵素基質的大約濃度。

5. Vmax 的意義：
a.在足夠的基質濃度下，一定量的酵素所能催化的最高反應速率，即為其
Vmax；要讓一個酵素達致其 Vmax，就要把基質量調至最高濃度。在比較不同
酵素的 Vmax 活性時，注意要以同樣莫耳數的酵素分子為基準。
b.單位時間內每莫耳酵素所能催化的基質數 (莫耳數)，稱為 turn over number 或
molecular activity，一般酵素約在 0.1~10,000 間 (每秒)，有大有小不等。 這是
當基質量極大於 Km 時 ([S] >> Km)，反應推向右邊，E + S → ES → E + P，
其 k3 成為決定因素，即為 turn over number，特標記為 kcat。
c.當基質量遠小於 Km 時 (Km >> [S] 則 [Et] = [E] 而 Km + [S] = Km)，則可以
由 M-M 公式導得：
Vmax [S] k3 [Et][S] k3 [E][S] kcat
vo ＝ ──── ＝ ──── ＝ ──── ＝ ── [E][S]
Km + [S] Km + [S] Km Km
反應速率成為 second order，由 [E] 及 [S] 兩項因素決定之。
而 kcat / Km 常數的大小則為重要指標，同時顯示酵素的催化效率及專一性。
d.瞭解上述的 Km 與 Vmax 後，重新回顧最早的酵素與基質反應式：
若把此式分成兩半，前半是 E + S → ES 由 k1 與 k2 主導；後半 ES → E + P
由 k3 主導。 則顯然 Vmax 是後半反應決定 (記得 Vmax = k3 [Et])，而 Km 則
大體上由前半反應所定。 因此整個酵素反應，是由這兩半反應所共同組成，前
半以 Km 來決定酵素與基質的親和度，後半反應以生成物的產生來決定最高反
應速率。注意 Km 的定義是 (k2 + k3) ÷ k1，故後半反應還是對 Km 有影響。

6. 酵素活性定義：
有兩種表示酵素活性的方式，請注意其定義不同，不要混淆。
a.活性單位：
酵素活性的表示方法通常使用 活性單位 (unit)︰ 即酵素 每分鐘 若催化 1
mmole 基質 的活性，即定義為一單位活性：注意同一酵素可能會有不同定義
方式的活性單位。
b.比活性：
每單位重量蛋白質 (mg) 中所含的酵素活性 (unit)，稱為 比活性 (specific
activity, unit/mg)；因酵素為活性分子，有時會失去活性，雖然蛋白質仍在，但
比活性會下降。

C. 雙基質反應：
雙基質反應 (Hexokinase 也是雙基質反應)
a.上述 S→P 催化反應，只有一種基質及一種生成物，稱為 Uni-Uni 反應。 但
事實上大多數酵素反應，有一個以上的基質，也可能有數個生成物，為多基質反
應。例如：
S1 ＋ S2 → P (Bi-Uni, 雙-單)
S → P1 ＋ P2 (Uni-Bi, 單-雙)
S1 ＋ S2 → P1 ＋ P2 (Bi-Bi, 雙-雙)
b.雙基質反應仍可適用於 M-M 公式，但 兩種基質的 Km 要分別測定；測 S1
的時候，反應中的 S2 濃度要飽和 (使 S2 成為非主導因子)，反之亦然。
c.Bi-Bi 反應中基質 (S1, S2) 及生成物 (P1, P2) 的進出次序有數種情形：
(1) Random：基質進入活性區並沒有一定次序，但兩個基質都要結合到酵素
後，才會開始進行反應。
(2) Ordered sequential： 基質依固定次序進入，然後生成物再依序出來。
(3) Ordered ping-pong： 依 [S1 進, P1 出; S2 進, P2 出] 次序，像是兩個
Uni-Uni 組成的；也像是打乒乓球一樣地一來一往。

5. 酵素的抑制：
酵素的抑制：可逆 vs 不可逆 (好像汽車的煞車系統)
A.酵素的抑制方式：
a.以抑制劑與酵素結合而導致抑制作用，這種結合是 可逆 或 不可逆 反應都
有。
b.很多生理或藥理上的作用，都是源自於抑制劑對酵素的作用，而使酵素的活性
降低，或者完全失去活性。如消炎的 磺胺藥，即是一種細菌酵素基質 (PABA)
的類似物，可抑制細菌葉酸的合成。
c.抑制劑與酵素產生非共價性結合，然後可以阻礙基質進入酵素活性區，或者改
變酵素構形而使其失活。
d.抑制酵素的機制，依 抑制劑 [I] 與 酵素 [E] 的結合方式，可以分成三種：
Competitive, non-competitive 及 uncompetitive； 由抑制劑對酵素動力學曲線所
造成的影響，即可得知是何種抑制方式。
B.不可逆的抑制：
不可逆性抑制劑會對酵素活性區上的主要胺基酸做 共價性修飾，因此酵素活性
通常被嚴重破壞，便無上述三種動力學抑制方式。
a.青黴素 (penicillin) 喬裝成基質，可與細菌的一種酵素發生不可逆的結合，此
酵素乃細菌細胞壁生合成的重要酵素，細菌無法正常生成細胞壁而死亡。 有點
像是分子版的『木馬屠城記』。
b.重金屬：Hg2+, Pb2+, Cd2+ 及砷化物等重金屬，非專一性地與 [E] 或 [ES] 結
合，取代原來酵素所需的金屬，而使酵素失去活性。
c.化學修飾劑： 某些化合物可以 專一性 地修飾特定胺基酸，除了可做為專一
性抑制劑外，也可用來檢定酵素活性區中，具有催化反應的胺基酸為何者。
d.蛋白酶及其抑制劑： 蛋白酶廣泛存在於細胞中，有其專一性抑制劑可控制其
生理活性，二者互相抗拮形成一調節控制網。目前極被重視的 HIV 蛋白酶及
其人工抑制劑，在醫藥研究上有很大的作用及影響。

6.酵素的催化機制：
酵素的催化機制可以化學反應逐步推得，與分子的構造也有相當大的關係。
A.酵素活性區 (active site)︰
酵素提供基質一個穩定的空間，有利於穩定其過渡狀態，並快速轉變成為生成物。
a.活性區： 是酵素與其基質 (或輔酶) 的結合區域，並在此進行催化反應。 活
性區通常是一凹陷的袋狀構造，水分子不易進入袋中。 活性區內的胺基酸，只
有那些具 反應性基團 (reactive group) 者，才直接參與催化反應；但各種胺基
酸都有可能參與結合基質。
b.酵素催化的 化學機制，通常是以下面三種基本方式進行反應：
(1) Bond strain：基質結合到酵素後，酵素的構形扭曲使基質分子內某共價鍵受力
斷裂。
(2) Acid-base：利用活性區內胺基酸基團或輔酶，可以放出或接受質子 (或電子)
的特性，對基質進行質子或電子的轉送。
(3) Orientation：活性區可提供基質適當的排列空間，使有利於反應進行。
以上三種方式或可同時發生，為 協同式 (concerted set)；亦可先後依序發生，稱
順序式 (sequential mechanism)。 以下兩個重要例子都是蛋白酶。
B.協同式催化機制︰
以 carboxypeptidase A (CPA, 羧肽酶) 為模型酵素說明之。
CPA 的分子量 34 kD，含 307 個胺基酸，有一雙硫鍵，及一個鋅離子，是一種
金屬蛋白。 CPA 可由胜汰的 C-端，依序切下外側胺基酸 (外切酶)，當胺基酸
的 R 基團為非極性者，較有利反應進行；而 carboxypeptidase B (CPB) 只切 C-
端為 Lys 或 Arg 者，二者專一性不同。
C.順序式催化機制︰
以 chymotrypsin (CT, 胰凝乳蛋白酶) 為代表，請找出課本的相關圖片。
a.分子構造： CT 的分子量為 25 kD，含三條胜肽，由兩個雙硫鍵連接，是 轉
譯後修飾 的產物；剛轉譯出來的完整胜肽鏈沒有活性，要在接近 N-端的 Arg
15 與 Ile 16 之間先斷開後，CT 才能活化。
b.催化活性： CT 可水解胜肽鏈上面的芳香族胺基酸 (Tyr, Phe, Trp) 或 Met (具
較大非極性基團者)，切開這些胺基酸 C-側的胜鍵，是一種 內切酶。
c.活性區： CT 大部分的極性胺基酸都露在分子外表，只有三個在活性區內，對
催化反應扮重要角色 (Ser 195, His 57, Asp 102)。三者以『電荷接力』形成 高
反應性 Ser：其 Ser 195-OH 基上的 H+ 被鄰近的 His 57 吸收，生成具有高
反應性的 -O-。
d.催化機制： Ser 上的高反應性 -O - 會攻擊基質胜肽上的 carbonyl 碳 (帶微正
電)，形成新的 C-O 鍵 (acylation 步驟)，同時斷開基質的胜鍵，先釋出一段 C-
側的胜肽；然後加水分解，破壞此 C-O 鍵 (deacylation 步驟)，釋出另一段 N-
側胜肽。以上兩個步驟，依次順序發生。
e.穩定過渡狀態： 除了 catalytic triad 可以有效催化之外，活性區也可以穩定催
化反應的中間過渡狀態；過渡狀態有相當高的負電荷，因此以活性區附近的 Gly
193 及 Ser 195 本身的 -N-H 與之產生氫鍵而中和之。
f.Ser 蛋白酶家族： 這種以 Ser 為催化主力的蛋白酶很多，統稱為 Serine 型蛋
白酶；除了 chymotrypsin 外，尚有許多如 trypsin (胰蛋白酶)、elastase (彈性蛋
白酶)。它們的催化機制相似，立體構形相當類似，且都有 [Ser-His-Asp] 接力
形式的催化機構。 但對基質的專一性不同，trypsin 的基質為鹼性胺基酸 (Lys,
Arg)，elastase 則只切 R 基團較小且不帶電荷者。
D 酵素的專一性：
酵素只與特定的基質結合，有很高的專一性，這是酵素重要特性之一。
（一）專一性結合區：
大部份酵素的活性區即有專一性辨識與結合基質能力，但 CT 另有一基質的辨
識區位在其活性區附近。
a.上述各 Ser 型蛋白酶的活性區除了有 [Ser-His-Asp] 催化中心外，附近還有一
個袋狀的基質結合區，可以辨別基質的種類 (驗明正身)，以便與正確的基質胺
基酸結合。
b.這種專一性結合是靠結合區上胺基酸 R 基團的 形狀、大小、極性 或 電荷 等
性質的契合，以 二級鍵 結合。 例如 chymotrypsin 的結合區中多含非極性的
胺基酸，故只能與非極性的芳香族胺基酸結合，催化這類胺基酸的水解。改變
結合區的重要胺基酸，即可改變專一性 (Science, 1992, 255: 1249)。
（二）專一性結合力量：
有兩大類構成因素，一種是形狀的互補，另一種是吸引力量。
a.分子間的 構形互補： 有如『鎖頭與鑰匙 lock & key』在形狀上的契合；但蛋
白質分子較有彈性，與基質結合後可誘導生成更契合的構形，稱為 induced
fit。造成這種構形互補的主要力量是 凡得瓦爾力，如 圖 5 中 A 與 B 兩分
子間的契合。
b.酵素與基質間會產生 吸引力量，是由兩者間的若干二級鍵所共同組成。
（三）立體專一性：
酵素分子如何辨認立體異構物？ 碳原子的原子軌道 是根本原因。
a.酵素可辨認基質的 立體不對稱性，只能對某一種立體異構物有催化反應 (如 L
或 D 型胺基酸之一)，而生成物也只有對應立體異構物之一。
b.基質分子中 不對稱碳 (sp3) 四個鍵結中的一點 (A) 固定，再以酵素的專一性
結合面接觸並確認 B-C-D 三點，就能分辨另一異構物的差異 (可能是 B-D-C)
而排斥之

7.酵素活性的調節：
酵素活性的調節，對細胞生理是極為重要的，因為細胞並不一定期望某酵素一直
保持在活性狀態。對酵素分子的共價或非共價性修飾，其調節活性的效果最為直
接而迅速；有些是可逆反應，也有部分是不可逆。以下面四種方式，加上抑制劑
的使用，細胞就可以自由控制酵素活性的高低。
A.蛋白質裂解：
不只是酵素，很多蛋白質都是利用降解來調節其活性或功能。
（一）酶原或前驅體(Zymogen or proenzyme)：
以蛋白質的裂解來調節酵素活性，該酵素在裂解後得以活化；也可以把完成任務
後的蛋白質分解掉，以免繼續進行著生理作用。
a.許多酵素 (或蛋白質) 剛由 mRNA 轉譯成蛋白質時，分子量較大而不具活
性，稱為 酶原 (zymogen)；在蛋白質則稱 前驅體 (precursor)。 酶原或前驅體
再經蛋白酶切開，或除去部分胜肽，才能成為具有活性的分子。 請研究下面數
例，注意 胰島素及腦啡是荷爾蒙而非酵素，而打有 * 號者乃表示具有活性者。
(1) 凝血酶： Prothrombin → Thrombin* → (凝血反應)
凝血機制 凝血對生物體是一件很嚴重的事，因為血液不能在血管內任意凝固。
因此細胞對誘發凝血的過程，控制得非常嚴格，是以一種叫做『梯瀑 cascade』
的方式，把一個信息慢慢放大；而在放大過程中，有許多管制點，可避免因假警
報誤導致凝血。上面的凝血酶生成是最後一步，凝血酶可以馬上誘發凝血。
(2) 胰島素： Preproinsulin → Proinsulin → Insulin*
轉譯後的 preproinsulin 先摺疊好以後，切除 N-端的一段胜肽成為 proinsulin，
然後再切除中央一段 C 胜肽後，才會有活性。
(3) 腦啡：合成一長條八元體的前驅體，再以一種類似 trypsin 的蛋白酶裂解成
單位腦啡，因此其產生也受到蛋白酶的控制。
b.許多破壞性酵素 (如蛋白酶) 先以酶原形式合成出來，以免在到達作用目標前
破壞其它蛋白質；也有以胞器或細胞膜隔離者，稱 區隔化 (compartmentation)。
Chymotrypsinogen → π-Chymotrypsin* → α-Chymotrypsin*
Chymotrypsinogen 分子的斷裂會使其構形改變，露出新的 N-端 Ile 16，這個新
的 -NH3+ 會吸引 Asp 194 的酸基，進而固定隔壁的 Ser 195，使其就定位，
並因此打開專一性結合區，轉變成為活性型。
c.這種酶原裂解的活化方式是不可逆的，因為被切掉的片段無法接回；因此細胞
以 抑制劑 (inhibitor) 來控制此類酵素。Trypsin 及其抑制劑為一範例，trypsin
被切開活化之後，只能用其專一性抑制劑去控制。
（二）蛋白酶：
越來越多的研究發現，蛋白酶在細胞內具有重要的生理功能。
a.依催化機制可分成四類： (1) 金屬蛋白酶 (2) Serine 蛋白酶 (3) Cysteine (或
Thio) 蛋白酶 (4) Aspartyl (或 Acid) 蛋白酶。
b.同一族的蛋白酶序列都具有類似的胺基酸序列及構形，尤其在活性區的保守性
胺基酸幾乎絕對不會改變 (如 Ser 蛋白酶的 catalytic triad)；其它序列則因 趨
異演化 而衍生出許多具有不同專一性的蛋白酶。 與蛋白酶構造完全無關的
acetylcholinesterase，則因 趨同演化 而具有類似 catalytic triad 的 ester 水解能
力。
c.類 似 核 酸 的 intron 與 exon ， 蛋 白 質 也 有 自 我 剪 接 的 現 象 ， 稱 intein 與
extein，是轉譯後的修飾作用；但此現象並不是很普遍。
（三）Ubiquitin-Proteasome 降解路徑：
細胞內蛋白質的降解，可能是一種調節性的生理作用；最近發現很多蛋白質，都
要先標上 ubiquitin 後，才以 proteasome 來進行降解。
a.Ubiquitin (泛素)：廣泛分布在動植物細胞中，同質性高而分子量小，可以連到
目標蛋白質分子的 Lys 胺基，作為被降解的標記 (ubiquitination)。目標蛋白質
的胺基酸序列上，通常在近 N-端有一定 信號序列 (destruction box)。
b.Proteasome (蛋白酶體)：是由很多較小單位分子所組成的巨大分子，主體看來
像是四個甜甜圈疊在一起，中央的孔洞可以容納目標蛋白質，並將其分解成胜
肽片段；目標蛋白質都要先標有 ubiquitin，水解後 ubiquitin 可以回收再次使
用。
B.磷酸化 (phosphorylation)：
磷酸化是非常廣泛且多樣的蛋白質修飾方式，在信息傳導上極為重要；越來越多
的重要生理現象，被發現是與蛋白質的磷酸化有關。而 肝糖磷解酶 (glycogen
phosphorylase, GP) 是磷酸化調控的典型例子，請仔細研究之。
a.蛋白質分子上某些胺基酸 (如 Ser, Thr, Tyr) 的 -OH 基團會被 蛋白質激酶
(protein kinase) 修飾，在其分子加上磷酸而致活化，此磷酸可再被 蛋白質磷酸
酶 (protein phosphatase) 去掉而失活；但亦有相反的例子。
b.蛋白質經磷酸化加上一個強負電基團，影響附近胜肽鏈的排列，造成蛋白質構
形改變，而使活性升高或降低。 除了磷酸化之外，蛋白質也可以接上 核苷酸 達
到活化。
c.最近發現有些磷酸化蛋白質也會進入上述 ubiquitin-proteasome 路徑，引發該
蛋白質的降解，以達調節目的。
C.非共價結合之信息傳導分子：
主要代表分子是 cAMP 及 calmodulin，都是信息傳導途徑的重要媒介，與上述
的磷酸化反應共同合作，組成有效的細胞調節網路。
（一）cAMP：
蛋白質激酶 之活性受到 cAMP 調節，原不具活性的激酶與 cAMP 結合後，會
釋出活化型的蛋白質激酶。
（二）Calmodulin (攜鈣素)：
當細胞內的鈣離子濃度改變時，會誘導許多生理反應，而 calmodulin 即為細胞
內鈣濃度的感應與作用分子； 它的分子 (17 kD) 上有四個鈣離子結合位置，與
鈣結合後會誘導其分子構形的改變，可與目標蛋白質結合，調節後者的活性。
（三）信息傳導路徑：
當荷爾蒙或細胞激素等胞外信息分子到達目標細胞，與胞膜上的專一性受體結合
後，就會引發該細胞的一連串反應，把胞外信息帶入細胞內，以啟動所要的生理
反應。細胞內的這些反應，相當複雜而有效，統稱為 信息傳導；由上面所述的
磷酸化及 cAMP 等小分子為主軸，加上目標酵素的活化，可分成幾個層次，各
層次由許多模組所構成。
下面整理出這幾個層次，並且舉出一個對應例。
Signal → Receptor → Transducer → Effector enzyme → Effector → Effect
對應的分子如下例：
Glucagon → Receptor → G-protein → Adenylate cyclase → cAMP → Kinase
這種傳導途徑有兩個特點，是細胞生理極為重要的手段：
a.放大作用 (amplification)：上述一層一層的傳導過程中，有些是酵素 (如 cyclase,
kinase)；而一個酵素分子若經活化，代表著將有大量的基質會被催化成生成物，
而生成物會再傳導給下一步反應，信息因此而被放大。這種方式，非常像真空
管的電流放大作用，也是上述 cascade 梯瀑式放大的一種。
b.彈性模組 (flexibility)：信息傳導的每一步代表一個層次，而每個層次可由各種
模組所組成，因此可以有不同彈性組合，以因應不同細胞的種種需要。
D.異位酶 (allosteric enzyme)：
有些酵素的活性，會被它下游的產物所調節，是為 迴饋控制 (feedback) 現象；
這類酵素的分子上，除了有活性區可與其基質結合外，還有可與其下游產物結合
的位置，稱為 調節區 (regulatory site)，這種酵素則稱為 異位酶。
（一）Aspartate transcarbamoylase (ATCase)：
ATCase 是典型的異位酶。
a.迴饋控制： ATCase 催化 aspartate 與 carbamoyl-P 連結成 carbamoyl aspartate
的反應，後者經代謝後生成 CTP； CTP 則回頭與 ATCase 上的 調節區 結
合，反而抑制 ATCase 的活性 (負迴饋)。 由於 ATCase 是這個反應鏈的起始
酵素，控制 ATCase 即可控制整條代謝路徑。
b.四級構造： ATCase 是由兩組 催化次體 (catalytic subunit, CCC) 以及三組 調
節次體 (regulatory subunits, RR) 組成；每組催化次體由三條蛋白質組成 (C, 34
kD)，每組調節次體由兩條蛋白質組成 (R, 17 kD)：
2 × (CCC) ＋ 3 × (RR) ＝ C6R6
每調節次體有一 效應物結合區，每催化次體有一 基質結合區。
c.S 型曲線： ATCase 的動力學結果顯示，以 vo 對基質 [Asp] 濃度直接作圖，
可得到一 S 型曲線 (sigmoidal curve)，而非典型的 M-M 單股雙曲線。 這是
合作式 (cooperative) 的 協力現象，表示基質與酵素之任何一個次體結合後，
可誘導改變酵素的分子構形，促進其它次體與基質間的結合能力 (positive
homotropic effect)。
d.分子開關： 此 S 曲線有一轉折點，基質在達到這個基質濃度後，酵素反應速
率急速上升。 可以將此轉折點看做酵素對其環境基質濃度的 感應點，當基質
濃度低於轉折點時酵素不太活動；而達到轉折點時，酵素便很快起動達到
Vmax。
e.協力現象： 若反應加入 CTP，則 S 型曲線下移 (但 Vmax 不變)，表示 CTP
會降低 ATCase 的活性 (negative heterotropic)； 而 ATP 則反可增強 ATCase
活性 (positive heterotropic)，但原 S 型曲線則變成一般的 M-M 雙曲線，協力
現象消失。

f.效應物 (effector)： ATP 與 CTP 均可影響此異位酶的活性，二者都是其代謝

上的相關產物，稱為 效應物 (effector)； 兩者在構造上與基質 Asp 均不相像，
故並非活性區的競爭者，而是結合在分子的其他位置 (異位調節區)，結合後會
影響酵素的分子構形，使酵素與基質的親和力下降，因而降低活性；而 ATP 與
CTP 乃互相競爭此一調節區。
（二）異位酶的作用模型：
當異位酶受到效應物的影響時，其分子可能會有不同的反應行為。
a.異位作用： 基質 (或效應物) 與異位酶的一個次體結合後，會使原來的 T (tense)
型次體，變成較易接受基質的 R (relaxed) 型，然後繼續影響其他次體的結合能
力，有兩種方式︰
(1) Concerted 協同式： 酵素分子的每個次體，在結合前後一起呈現 T 或 R
型，保持著對稱性；若酵素為二元體，則為 RR 或 TT，而無 RT 型。 效應物
的結合也是同樣情形，抑制劑會使酵素固定在 TT 型，活化劑使成為 RR 型；
此二者同時都失去協力現象。
(2) Sequential 順序式： 酵素的每個次體各自與基質 (或效應物) 結合後，個別
由 T 型轉成 R 型，並不影響其他次體的構形 (仍為 T 型)，但對其他次體與基
質的親和力，可能有正或負影響。 依基質加入順序，分子構形變化為 TT → RT
→ RR。
b.兩種效應方式： 基質與異位酶的結合，通常是協同式；一個次體與基質結合
後，誘導所有次體都轉成 R 型，使其他次體的活性區更易接受基質。如此影響
其他次體上同種結合區者 (活性區→活性區) 稱 homotropic (同質效應)。 而效
應物與異位酶的結合多為順序式，且會影響其他次體與基質的結合能力；此種
不同種結合區之間相互影響 (調節區→活性區) 則稱為 heterotropic (異質效
應)。
8.細胞代謝與酵素調控：
酵素與細胞代謝極為密切，因為所有的代謝路徑，全是由酵素與其基質所組成
的；此外，還要加上對酵素活性的調節控制，以便使得細胞正常運作。

A.細胞代謝途徑：
細胞經同化作用合成所需分子，由異化代謝消耗分子、取得能量，全由酵素所控
制。
（一）代謝調控原則：
控制酵素即可控制代謝路徑，控制代謝的大原則如下各點。
a.一個基因一個酵素： 細胞的代謝途徑極為複雜，但並非細胞所有的代謝路徑
都在進行；進行中的每一步代謝，都由某一種酵素負責催化反應，而每一個酵
素分子都是由一個對應基因所轉錄。因此某基因的開啟或關閉，到最後可能會
影響代謝途徑的進行。
b.速率決定步驟： 因此控制該酵素的 活性 或 生合成量，即可控制該步驟反應
的快慢；若此反應為某一系列代謝路徑的 速率決定步驟 (rate-limiting step)，則
可控制這整條代謝途徑。
c.可逆或不可逆： 大部份酵素反應是 可逆 的，有時為了使反應保持在某一方
向，則成為不可逆反應；不可逆反應大多與消耗 ATP 的反應耦合。
d.代謝路徑可互通： 許多代謝路徑間若有共同的中間物，則可互通，也可能有
旁支或小路相連；因此若某條重要路徑失效，細胞通常不會立刻死亡，而會互
補保持一種動態的穩定狀況。
（二）異化代謝途徑鳥瞰：
以生物分子而言，異化代謝路徑可分成三個層次，有計畫地把巨分子逐步分解，
最後得到能量；其中最主要的是一條 糖解作用 (glycolysis)。
a.Stage 1: 巨分子 (蛋白質, 多糖, 脂質) 消化成單元小分子 (胺基酸、單糖、脂
肪酸)，可說就是消化作用。
b.Stage 2: 單元小分子再分解成更小的 acetyl CoA，初步得到一部份的能量。
c.Stage 3: Acetyl CoA 經過氧化磷酸化反應得到大量能量，分解成 CO2 及水。
（三）糖類中心代謝途徑：
由糖解作用到氧化磷酸化反應，是細胞最主要的一條代謝大道；學習細胞的代謝
途徑，可以此為中心，其它各類大小分子的代謝都可匯入此中心。
a.糖解作用： 把葡萄糖分解成 pyruvate → acetyl CoA (所有代謝分子的焦點)。
b.檸檬酸循環：在粒線體中把 acetyl CoA 再分解成 CO2，產生 NADH。
c.氧化磷酸化反應： NADH 轉換成 ATP 並生成水。
C.代謝途徑中酵素的調控：
細胞如何控制代謝途徑上的各種酵素？ 可以針對酵素本身進行修飾，或者控制
其基因的表現。而荷爾蒙或細胞激素，是在細胞間傳遞長短程控制指令的信息分
子。
（一）基因表現的調控：
酵素在不同細胞內的表現量可能不同，同一細胞內的各種酵素量也不同。
a.酵素的 生合成 受到其基因的控制 (DNA→mRNA→protein)，因此基因的開或
關，或其表現程度，會影響該酵素在細胞內的量，進而影響該酵素所控制的代
謝反應，是為 基因表現 (gene expression)。 一個表現中的基因，應該有大量
mRNA 轉錄出來，但有大量 mRNA 卻不一定產生大量酵素。
b.一條代謝路徑的起始基質，可能誘導關鍵酵素基因的表現，稱為 induction；此
代謝的最終產物，也可能抑制酵素表現，稱為 repression。 基因操縱子 (operon)
即是一例，乳糖能夠去除 repressor 與 lac operon 之結合，而使基因開動。
（二）酵素活性調節：
已在上一節詳述，再整理成共價及非共價修飾兩大類。是針對酵素分子所進
行蛋白質層次的修飾調控，與上述之基因調控方式不同。
a.非共價修飾：
使用 cAMP 或正負迴饋的 效應物 等方式，以非共價方式修飾酵素活性，此多
為可逆性的調控。勿把異位酶與上述操縱子的調控方式混為一談，兩者都可被
基質活化，但前者是修飾酵素本身，而後者是影響基因的表現。
b.共價修飾：
以磷酸化或蛋白質水解(zymogen，酶原)的方式，來增強或降低酵素活性。 多為
cascade 式的連鎖代謝反應，cascade 連鎖反應會放大 (amplify) 某條代謝路徑
的活性。除了正向的以生合成增加酵素量之外，蛋白質的降解也是一項重要的
調控方式，並以蛋白酶或 ubiquitin 配合 proteasome 進行降解，以除去此酵素。
（三）激素調控：
細胞與細胞之間如何傳遞信息？ 這是一個極有趣而且重要的問題。
a.荷爾蒙 (如 insulin) 傳遞 長程 生理指令，經由血液傳送並與目標細胞接觸，
結合到細胞膜上的接受器 (receptor)，可引發一系列的信息傳導反應，把『啟動』
的指令傳入細胞內，藉著蛋白質激酶或磷酸酶等，活化某些關鍵酵素，開始進
行該細胞所預設的功能 (如分解肝糖)，以應身體所需。
b.細胞激素 (cytokine) 則多在免疫細胞之間傳遞 短程 的信息，可刺激局部的細
胞增生；也是利用信息傳導路徑，來啟動細胞內的各種生理功能。
（四）細胞空間的效用：
利用酵素或基質在空間上的隔離或聚集，是一有效控制方法。
a. 胞器隔離：
真核細胞內有許多 隔間 (compartmentation)，形成其胞器的隔離空間；在胞
器內可聚集較濃的特定酵素及基質，以便有效進行催化反應，或避免有害的
 副作用。 例如葡萄糖中心代謝途徑中，檸檬酸循環及氧化磷酸化反應是在粒
線體中進行的。
b. 酵素複合體：
幾個連續反應的酵素，可組合在一起成為複合體；則某酵素的生成物，可馬
上被下一個酵素用為反應物，降低擴散碰撞所需時間。 例如葡萄糖中心代謝
途徑中的 pyruvate dehydrogenase (催化 pyruvate → acetyl CoA) 是由三種酵
素組成的複合體。
c. 膜上酵素群：
一群連續反應的酵素，也可以一起附在細胞膜上，除了可加速反應速率外，
其作用也可能與細胞膜有關。 例如 葡萄糖中心代謝途徑 中，氧化磷酸化反
應酵素群 是附在粒線體的內層膜上，並利用膜內外的質子濃度差異來產生
ATP。

8.酵素在生物技術上的應用：
酵素在現代生物技術的研究或應用上，是一個非常重要的範疇。雖然基因群殖等
分子生物的研究蓬勃，但基因表現的產物仍是蛋白質或酵素；另外，在基因操作
所應用到的核酸剪接工具，幾乎全部是酵素。 以下列舉部份目前生物技術研究
常見的一些應用，以及最近的重要發展。
A.酵素免疫分析法 (ELISA)︰
抗體可用來偵測其專一性抗原，而將抗體連接以酵素，可做為追蹤或定量的標幟；
通常把免疫試劑之一的抗體固定在 固定相 (solid phase)，以利沖洗分離，稱為
ELISA (enzyme linked immunosorbent assay)。
B.固定化酵素及酵素電極：
酵素經固定後有許多好處。
a.用物理或化學方法把酵素固定到固相擔體上，比一般使用的溶態酵素有以下優
點：
(1) 酵素 可回收 重複使用，較為經濟。
(2) 固相與液相的 分離方便，使用上速度快而分離完全，有助於自動化。
(3) 許多酵素是附在細胞膜上，固定化酵素可 模擬 細胞內酵素的實際環境。
b.利用上述酵素的固定化，把酵素固定在半透性薄膜上，連接到電極，偵測反應
進行的結果 (例如 pH 的改變)，可作為酵素反應的自動化偵測工具。

C.蛋白質工程及人造酵素︰
以基因重組或其它方法，可以大量生產某種酵素，也能改變酵素的催化特性。
a.分子群殖 molecular cloning：
cDNA 包含完整的蛋白質轉譯訊息，若把某蛋白的 cDNA 插在表現載体中 (如
某質體)，則宿主可能表現此段轉殖基因，而生合成此蛋白質。若在此 DNA 接
 上另一種已知的酵素基因 (如 luciferase 或 GUS)，則表現出來的蛋白質是二
者的連結體，稱為 融合蛋白質 (fusion protein)；而此酵素的活性可做為追蹤之
用，稱為 reporter。
b.蛋白質工程：
若能改變酵素活性區的胺基酸，則可能改變酵素的活性，或是其專一性。 通常
先研究並預測改變其活性區某胺基酸後，可能引起的變化；再以 人工定點突變
(site-directed mutagenesis) 改變某核苷酸，然後以分子群殖操作表現該突變蛋白
質。
c.人造酵素：
酵素的活性區通常包含數個極性胺基酸，若在人造的分子骨架上，模仿活性區
的幾何位置，接上這些胺基酸，則可能得到具有催化作用的人造分子。
d .Abzyme (催化性抗體)：
若能得知酵素催化反應過程中，其基質轉換為產物的 過渡狀態 物質，以此物
質或其類似物作為抗原進行免疫，則所得到的抗體，可能具有催化能力。 但其
催化效率，遠不及自然酵素，通常只有千分之一的效果。 最主要原因在於酵素
的催化區是一凹陷口袋，可隔離外界干擾，提供最佳環境穩定過渡狀態；而
abzyme 的結合區較淺，無法十分有效地隔離並穩定過渡狀態 (Nature 1996, 383:
23-24)。

D. Proteome 蛋白質體：
分子生物學革新了整個生物學的觀念，也將會改變生物化學中酵素的研究方法。
（一）Genome project 基因體計畫：
二十一世紀的大事之一，是人類將可解讀出自身染色體內所有 DNA 的序列，此
一大計畫即稱為 Human Genome Project，由發現 DNA double helix 之一的 J.D.
Watson 所主持。 其他重要的植物或細菌，有很多也已經開始進行，甚至已經解
讀完成。
（二）Proteome 蛋白質體：
a.一旦解出某種生物的染色體 DNA 序列，接著有許多工作可以進行，其中最有
趣的是可以馬上把這些序列翻成將表現出來的蛋白質，如此我們就可以得知
某生物細胞內可能含有的全體蛋白質，稱之為 proteome 蛋白質體。
b.生物細胞內的蛋白質體中，當然只有一部份是酵素，但由細胞內所含有的酵素
種類，即可拼出該細胞可能的代謝途徑；由這些代謝途徑，就可以推出某細
胞是如何運作。
c.這種研究方式，與傳統的生物化學或生理學實在相當不同，要靠完整的資料庫
與功能強大的電腦軟體，是 生物資訊學 (bioinformatics) 的主要範疇。
Nucleotides and nucleic acids
The basics of nucleotides and nucleic acids
1. A nucleotide consists of a nitrogenous base, a pentose sugar, and phosphate
group(s).
2. Phosphodiester bonds link successive nucleotides in nucleotides.
3. The differences between DNA and RNA in structure:
(1). DNA: A, T, C, G; RNA: A, U, C, G.
(2). DNA: pentose-2-H; RNA: pentose-2-OH.
4. The two strands of DNA is antiparallel and complementary. That means, if the
sequence of one strand is 5GAACTACT3, then the sequence of the other strand is
5AGTAGTTC3.
5. In all cellular DNA, A=T, G=C. That is A＋G= T＋C.
6. The products of enzymatic or alkaline hydrolysis of RNA are
nucleoside 2-monophosphate, nucleoside 3-monophosphate,
nucleoside 5-monophosphate, nucleoside 2, 3-cyclic monophosphate.
7. DNA exists in A, B, and Z structural forms. The B form is the most stable
structure under physiological conditions.
8. The major groove is barely apparent in Z-form. The left-handed helix in Z-form,
the purines in syn form, alternating with pyrimidines in the anti form (CG rich
region). These Z-DNA tracts may play a role in regulating the expression of
some genes or in genetic recombination.
9. H-DNA triple helix contain two pyrimidine and one purine strands.
Nucleic acid chemistry
1. The higher its GC content, the higher the melting point (tm) of the DNA.
2. The mutations of DNA:
(1). Deamination: C→U, 5-methylcytosine→T.
(2). Depurination
(3). Formation of pyrimidine dimers (thymine dimer) induced by UV.
3. DNA methylation:
(1). A & C are methylated more often than G & T.
(2). 2. E. coli has two prominent methylation systems: defense system & repair
system.
(3). In eukaryotic cell, about 5% of C in DNA are methylated.
(4). Methylation is most common at CpG sequences.
(5). Methylation suppresses the migration of transposon.
(6). 5-methylcytosine in repetitive CpG sequences markedly increases the
segment of DNA to the Z form.
4. DNA sequencing: template, primer, dNTP, DNA polymerase, and ddNTP are
necessary.
DNA-based information technologies
DNA cloning
1. DNA cloning: restriction endonuclease: 4-6 bases. Sticky end & blunt end.
DNA ligase
Plasmids (vectors): foreign DNA (＜15kb) insert in the polylinker
region. Selectable markers:
Antibiotics-resistance genes, LacZ gene
(blue-white).
Transformation: Heat shock: 42℃, 90 sec.
Electroporation: high-voltage pulse.
Microinjection
Viral vector
2. Bacteriophage λ vectors permit the cloning of DNA fragments of up to 23,000
bp. Bacterial artificial chromosome (BAC) carries 100-300 kb DNA
fragment.
Yeast artificial chromosome (YAC) carries up to 2000 kb DNA fragment.
3. A powerful approach, site-directed mutagenesis, is used to study protein
structure and function changes the amino acids sequence of a protein by altering
the DNA sequence of the cloned gene.

From genes to genomes

1. Cell containing particular DNA sequence can be identified by DNA
hybridization method using probe.
2. Genomic DNA segments can be organized in libraries, such as genomic
libraries & cDNA libraries. cDNA libraries constructed from the mRNA.
3. The polymerase chain reaction (PCR) can be used to amplify selected DNA
segments from an entire genomic DNA.
4. SNP (Single Nucleotide Polymorphism): Every individual has ~0.1% of the
genome that is different. In average, every 1 kb has a SNP can be used as a
marker on the genome.

From genomes to proteomes

1. Two-dimensional gel electrophoresis: allows the separation and display of up to
1000 different proteins on a single gel. Compare the proteomes between
different tissues, different stages of development, or different treatments.
2. Microarrays (chips): DNA or protein chips. DNA segments (antibodies) from
known genes, a few dozen to hundreds of nucleotides long, are amplify by PCR
 and placed on a solid surface.
3. The yeast two-hybrid system: A sophisticated genetic approach to defining
protein-protein interactions is based on the properties of the Gal4 protein
(transcription factor), which activates transcription of certain genes in yeast.
Principles of metabolic regulation: glucose and glycogen
Glycogen breakdown
1. The terminal glucose is removed from nonreducing end by glycogen
phosphorylase.
2. The debranching enzyme possesses two activities, (α1→6) to (α1→4)
glucantransferase and (α1→6) glucosidase. The glucantransferase shift a block
of three glucose residues from the branch to a nearby nonreducing end which is
1→4 linkage. Then the single glucose residue remaining at the branch point is
cleaved by 1→6 glucosidase activity of the debranching enzyme.
3. The product is glucose 1-phosphate, which is converted to glucose 6-phosphate
by phosphoglucomutase.
4. Glucose 6-phosphate in skeletal muscle enters glyoclysis serve as an energy
source to support muscle contraction.
5. Glucose 6-phosphate in liver is converted to glucose by glucose 6-phosphatase to
increase blood glucose level.

Glycogen synthesis
1. The glucose must be activated as UDP-glucose before the glycogen synthesis.
2. A primer protein, glycogenin, is necessary to initiate a glycogen synthesis.
3. When the first UDP-glucose is bound to glycogenin, glycogen synthase starts the
synthesis process.
4. The branch points of glycogen are formed by amylo (α1→4) to (α1→6)
transglycosylase.

Regulation of metabolic pathways

1. Living cells maintain a dynamic steady state — homeostasis!
2. About 4,000 regulatory genes (~12% of all genes)
different time scales (from seconds to days)
different sensitivities
different regulatory mechanisms overlap
3. Cell is more sensitive to [AMP] than [ATP].

Reduced nutrient supply Glycolysis

[AMP] ↑
Increased exercise Fatty acid oxidation
Coordiated regulation of glycolysis and gluconeogenesis
1. The glycolysis is regulated at the levels of hexokinase, phosphofructokinase-1
(PFK-1), and pyruvate kinase.
2. The gluconeogenesis is regulated at the levels of pyruvate carboxylase,
FBPase-1.
3. Hexokinase IV is sequestered in the nucleus of the hepatocyte, but is released
when the cytosolic glucose concentration rises.
4. Hexokinase IV ( compared with Hexokinase I-III)：
(1). High Km
(2). Inhibited by the binding of a regulatory protein (in nucleus)
(3). Not inhibited by G-6-P.
5. PFK-1 is inhibited by ATP, and activated by fructose 2,6-bisphosphate.
6. Pyruvate kinase is inhibited by ATP and cAMP-dependent phosphorylation.
7. Glucagon or epinephrine rises [cAMP], which decreases [fructose
2,6-bisphosphate] through inactivates PFK-2 and activates FBPase-2.
8. Glycogen phosphorylase is activated by glucagon or epinephrine, which rises
[cAMP] and activate PKA. PKA makes glycogen phosphorylase from b form
(inactive) to a form (active).
9. Insulin stimulates GSK3 phosphorylation which makes GSK3 inactivated. The
result lets the glycogen synthase remain in active a form.
10. Insulin stimulates the synthesis of hexokinase II and IV, PFK-1, and pyruvate
kinase to increase the glycolysis. Insulin increase the activity of glycogen
synthase to stimulate glycogen synthesis.
11. In liver, glucagon stimulates glycogen breakdown and gluconeogenesis, while
blocking glycolysis.
12. In muscle, epinephrine stimulates glycogen breakdown and glycolysis,
providing ATP to support contraction.
The citric acid cycle
Production of acetyl-CoA (activated acetate)
1. Glycolysis occurs in cytosol. Citric acid cycle occurs in mitochondial matrix.
Oxidative phosphorylation occurs in the inner membrane of mitochondria.
2. Oxidative decarboxylation of pyruvate to acetyl-CoA by the pyruvate
dehydrogenase complex (PDH complex).
3. Pyruvate is oxidized to acetyl-CoA and CO2 by PDH complex.
4. Pyruvate dehydrogenase complex is composed of three enzymes: pyruvate
dehydrogenase (cofactor TPP), dihydrolipoyl transacetylase (with lipoyl
group, cofactor CoA), dihydrolipoyl dehydrogenase (cofactors FAD, NAD).
5. The mechanism of PDH complex reacts as a substrate channeling model
(P605).

Reactions of the citric acid cycle

1. The citric acid cycle (Krebs cycle TCA cycle) oxidized the compounds derived
from carbohydrates, fats, and proteins, to CO2, which is consist with ATP
production.
2. 30-32 ATP produced per glucose oxidation.
3. Some anaerobic microorganisms use the intermediates of TCA cycle as a
source of biosynthetic precursors (amino acids, nucleotides, heme etc.), not
energy. They lack the α-ketoglutarate dehydrogenase, a metabolic enzyme in
TCA cycle.
4. Three anaplerotic reactions（代謝物回補反應） replenish citric acid cycle
intermediates: PEP carboxylase, PEP carboxykinase, malic enzyme.
Regulation of the citric acid cycle

High［ATP］/［ATP］or［NADH］/［NAD+］ratios inhibit the reactions above.

The glyoxylate cycle

1. The glyoxylate cycle is active in the germinating seeds of some plants and in
certain microorganisms
2. The pathway takes place in glyoxylsomes in seedlings. It uses two molecules of
acetyl-CoA, and produces one molecule succinate per cycle. No CO2 produced.
3. Two additional enzymes in plant are needed: isocitrate lyase and malate
synthase.
4. The glyoxylate cycle converses fat storage in seed to glucose for energy source
purpose.
Principles of Bioenergetics
First Law of Thermodynamics
1. The law of conservation of energy
2. Energy cannot be created or destroyed, but only converted to other forms
3. Enthalpy (H): description of change of heat content during biological reactions
a. dependent upon electronic internal energies
b. ΔH < 0 indicate favored reactions

Reversible and Irreversible Reactions

1. Reversible reactions - proceeds slowly - in the equilibrium state
2. Irreversible reactions - spontaneous or favorable processes.

Second Law of Thermodynamics

1. The universe tends to the greater disorder
2. Entropy (S): measurement of randomness
a. dependent upon translational, vibrational and rotational internal energies
b. ΔS > 0 indicate favored reactions

Gibbs Free Energy

1. A system tends toward the highest entropy and the lowest enthalpy
2. The net driving force in a reaction is the free energy change:
ΔG = ΔH - TΔS
3. Favorable reactions: ΔH < 0 and ΔS > 0 ---> ΔG < 0
4. Exergonic reactions: ΔG < 0 (thermodynamically favorable reactions)
5. Endergonic reactions: ΔG > 0
6. At equilibrium: ΔG = 0

Coupled Reactions
1. A spontaneous reaction may drive a non-spontaneous reaction
2. Standard free-energy changes of coupled reactions are additive
3. High energy compounds contain high energy phosphate bonds -> hydrolysis
a. Resonance stabilization of the phosphate products
b. Increased hydration of the products
c. Electrostatic repulsion of the products
d. Resonance stabilization of products
e. Proton release in buffered solutions
Oxidative Phosphorylation
1. NADH from TCA cycle activity can be used for reductive biosynthesis
2. the reducing potential of mitochondrial NADH is most often used to supply the
energy for ATP synthesis via oxidative phosphorylation
3. Oxidation of NADH with phosphorylation of ADP to form ATP are processes
supported by the mitochondrial electron transport assembly and ATP synthase

Principals of Reduction/Oxidation (Redox) Reactions

1. Redox reactions = transfer of electrons from one chemical species to another
2. The oxidized/reduced form is referred to as an electrochemical half cell
3. Coupled electrochemical half cells have the thermodynamic properties of other
coupled chemical reactions
4. a coupled redox reaction is the oxidation of NADH by the electron transport
chain:
NADH + (1/2)O2 + H+ --> NAD+ + H2O
5. standard biological reduction potential is -52.6 kcal/mole
6. NADH oxidation has the potential for driving the ATP synthesis (ADP + Pi -->
ATP) which is +7.3kcal/mole
7. 3 moles of ATP generated per mole of NADH
8. 2 moles of ATP generated per mole of FADH2

Energy from Cytosolic NADH

cytosolic NADH oxidized via the electron transport system
1. yields 2ATP if by the glycerol phosphate shuttle
2. yields 3ATPs if via the malate aspartate shuttle
Glycolysis, Gluconeogenesis, and the Pentose Phosphate Pathway
Glucose
1. rich energy - oxidation to CO2 and H2O - ΔG’0 = -2,840 kJ/mol.
2. major fates:
(1). storage as polysaccharide or as sucrose
(2). oxidation – glycolysis – 3 carbon pyruvate – ATP & metabolic intermediates
(3). oxidation – pentose phosphate pathway – ribose-5-phosphate – nucleic acid
synthesis & NADPH reductive biosynthesis processes
Glycolysis
1. ATP generation: energy source in RBC & contracting skeletal muscle
2. precursors for triglyceride and fatty acid biosynthesis in adipose tissue and liver
3. precursors for synthesis of amino acids and 5-carbon sugars
Pyruvate
1. aerobic conditions
(1). oxidized -> CO2, + acetyl for acetyl-coenzyme A
(2). acetyl group further oxidized completely to CO2 by the citric acid cycle
(3). electrons passed to O2 in the mitochondrion, to form H2O
(4). energy from the electron-transfer reactions drives ATP synthesis
2. lactic acid fermentation
(1). contracting skeletal muscle - low oxygen conditions (hypoxia)
(2). catalyzed by lactate dehydrogenase (LDH)
(3). pyruvate reduced to lactate accepting electrons from NADH
(4). NAD+ regenerated for glycolysis
3. ethanol fermentation: pyruvate is converted under hypoxic or anaerobic conditions
into ethanol and CO2

Glucose Oxidation
1. net production of two moles each of ATP and NADH
Glucose + 2ADP + 2NAD+ + 2Pi --> 2Pyruvate + 2ATP + 2NADH + 2H+
2. NADH for mitochondrial ATP synthesis
(1). glycerol phosphate shuttle -> 2ATP
(2). malate-aspartate shuttle -> 3ATP
3. 1Glucose -> 2Pyruvate + 6 or 8 ATP
4. 2Pyruvate oxidation ---> TCA cycle -> 30ATP
5. Total yield 1Glucose ---> CO2 and H2O + 36 or 38 ATP
The two phases of glycolysis
1. chemical priming phase: 2ATP used from Glc to F1,6BP
2. energy-yielding phase: 4ATP + 2NADH produce from F1,6BP to pyruvate

The Hexokinase Reaction

1. The ATP-dependent phosphorylation of glucose to G6P by hexokinase
2. nonionic glucose into an anion
3. biologically inert glucose becomes activated
4. Type IV isozyme of hexokinase is glucokinase found in liver and kidney
5. Hexokinase activity can be inhibited by G6P accumulation, whereas glucokinases
are not. Excess glucose Æ accumulation in liver; Energy needed Æ peripheral
glucose utilization

PFK-1
1. rate-limiting step of glycolysis
2. F-1,6BP hydrolytic enzyme in the same cell compartment
3. highly regulated

Phosphoglycerate Kinase
1. only reaction of glycolysis or gluconeogenesis that involves ATP and yet is
reversible under normal cell conditions
2. 2,3BPG is an important regulator of hemoglobin's affinity for oxygen

Pyruvate Kinase
1. strongly exergonic reaction
2. the high-energy phosphate of PEP is conserved as ATP

Lactate Metabolism
1. lactate diffuses and transported to highly aerobic tissues cardiac muscle and liver
2. lactate is oxidized to pyruvate by LDH and pyruvate further oxidized in TCA
cycle
3. If high energy level in cells, carbons pyruvate proceeds to gluconeogenesis

Ethanol Metabolism
1. Hepatocytes -> alcohol dehydrogenase (ADH) -> ethanol to acetylaldehyde
2. Acetaldehyde forms adducts with proteins, nucleic acids and other compounds
resulting toxic side effects with alcohol consumption
3. malate-aspartate shuttle and glycerol-phosphate shuttle
 4. activity of ADH vs Acetylaldehyde dehydrogenase Æimbalance NADH/NAD+

Regulation of Glycolysis
1. allosterically controlled hexokinase, PFK-1 and PK
2. Regulation of hexokinase not the major control point in glycolysis because G6P
are mostly derived from the breakdown of glycogen
3. Regulation of PK is important for reversing glycolysis (i.e. gluconeogenesis)
when ATP is high
4. The rate limiting step in glycolysis is the reaction catalyzed by PFK-1
5. PFK-1 is a tetrameric enzyme and ATP is both a substrate and an allosteric
inhibitor of PFK-1
6. Each subunit has two ATP binding sites, a substrate site and an inhibitor site
7. F6P is the other substrate for PFK-1
8. The most important allosteric regulator of both glycolysis and gluconeogenesis is
fructose 2,6-bisphosphate
9. F2,6BP, which is not an intermediate in glycolysis or in gluconeogenesis
10. Regulation of glycolysis also occurs at the step catalyzed by pyruvate kinase,
(PK)
11. PK is inhibited by ATP and acetyl-CoA and is activated by F1,6BP
12. The major allosteric effectors are F1,6BP, which stimulates PK activity by
decreasing its Km(app) for PEP, and for the negative effector, ATP
13. Muscle PK (M-type) is not regulated by the same mechanisms as the liver
enzyme.
14. differential regulation is that hormones such as glucagon and epinephrine favor
liver gluconeogenesis by inhibiting liver glycolysis, while at the same time,
muscle glycolysis can proceed in accord with needs directed by intracellular
condition

Entry of Non-Glucose Carbons into Glycolysis

1. Fructose Metabolism
(1). Muscle contains only hexokinase can phosphorylate fructose to F6P
(2). Liver which contains mostly glucokinase
(3). Hepatic fructose is phosphorylated by fructokinase yielding F1P
(4). F1P used by aldolase generates DHAP and glyceraldehydes
2. Galactose Metabolism
(1). Galactose, metabolized from the milk sugar, lactose
(2). enters glycolysis by its conversion to glucose-1-phosphate (G1P)
(3). galactose by galactokinase yields Gal-1-P
 (4). Epimerization of Gal-1-P to G1P from UDP-glucose catalyzed by
galactose-1-phosphate uridyl transferase to UDP-galactose and G1P
(5). UDP-gal is epimerized to UDP-glu by UDP-galactose-4 epimerase
(6). UDP portion is exchanged for phosphate generating glucose-1-phosphate
which then is converted to G6P by phosphoglucose mutase.
1. Mannose Metabolism
(1). mannose wis phosphorylated by hexokinase to generat M6P
(2). M6P is converted to F6P by phosphomannose isomerase
(3). or converted to G6P by the gluconeogenic pathway of hepatocytes

4. Glycerol Metabolism
(1). glycerol from adipose tissue
(2). backbone for the triacylglycerols
(3). transported to the liver and phosphorylated by glycerol kinase yielding
glycerol-3-phosphate
(4). Glycerol-3-phosphate is oxidized to DHAP by glycerol-3-phosphate
dehydrogenase
(5). DHAP then enters the glycolysis if the liver cell needs energy
(6). more likely enter the gluconeogenesis pathway in order for the liver to
produce glucose for use by the rest of the body.
Gluconeogenesis
1. biosynthesis of new glucose, (i.e. not glucose from glycogen)
2. reversal of glycolysis
3. 3 reactions of glycolysis with large negative ΔG0’ are bypassed during
gluconeogenesis by using different enzymes: (pyruvate kinase, PFK-1, and
hexokinase) -> (pyruvate carboxylase, PEP carboxykinase,
fructose-1,6-biphosphatase, glucose-6-phosphatase)
4. Bypass 1: Pyruvate to PEP
Pyruvate + ATP + GTP + H2O -> PEP + ADP + GDP + Pi + 2H+
5. Bypass 2: Fructose-1,6-bisphosphate to Fructose-6-phosphate
6. Bypass 3: Glucose-6-phosphate (G6P) to Glucose

Substrates for Gluconeogenesis

1. Lactate
(1). predominate source of carbon atoms for glucose synthesis by
gluconeogenesis.
(2). lactate released to the blood stream and transported to the liver and converted
to glucose
 (3). glucose is then returned to the blood for use by muscle as an energy source
and to replenish glycogen stores Æthe Cori cycle
2. Pyruvate
(1). Pyruvate generated in muscle and other peripheral tissues
(2). transaminated to alanine and is returned to the liver for gluconeogenesis
(3). The transamination reaction = glucose-alanine cycle =indirect mechanism for
muscle to eliminate nitrogen while replenishing its energy supply Æ amino
nitrogen is converted to urea in the urea cycle and excreted by the kidneys
3. Amino Acids
(1). All 20 amino acids, excepting leu and lys Æ TCA cycle intermediates
(2). carbon skeletons of the amino acids Æ those in oxaloacetate and
subsequently into pyruvate.
(3). When glycogen are depleted, in muscle during exertion and liver during
fasting, catabolism of muscle proteins to amino acids are major source of
carbon for maintenance of blood glucose levels.
4. Glycerol
(1). The glycerol backbone of lipids
(2). phosphorylation to glycerol-3-phosphate by glycerol kinase and
dehydrogenation to dihydroxyacetone phosphate (DHAP) by
glyceraldehyde-3-phosphate dehydrogenase(G3PDH)
(3). the glycerol-phosphate shuttle
(4). triacylgycerols of adipose tissue
5. Propionate
(1). propionyl-CoA
(2). succinyl-CoA
(3). only has quantitative significance in ruminants.

Regulation of Gluconeogenesis
1. negative effectors of glycolysis are positive effectors of gluconeogenesis
2. Regulation of the activity of PFK-1 and F1,6BPase
3. F2,6BP which is a powerful negative allosteric effector of F1,6Bpase activity
4. The level of F2,6BP will decline in hepatocytes in response to glucagon
stimulation as well as stimulation by catecholamines.
5. Each of these signals is elicited through activation of cAMP-dependent protein
kinase (PKA).
6. One substrate for PKA is PFK-2, the bifunctional enzyme responsible for the
synthesis and hydrolysis of F2,6BP. When PFK-2 is phosphorylated by PKA it
acts as a phosphatase leading to the dephosphorylation of F2,6BP with a
 concomitant increase in F1,6Bpase activity and a decrease in PFK-1 activity
7. F1,6Bpase activity is regulated by the ATP/ADP ratio. When this is high,
gluconeogenesis can proceed maximally.
8. Gluconeogenesis is also controlled at the level of the pyruvate to PEP bypass. The
hepatic signals elicited by glucagon or epinephrine lead to phosphorylation and
inactivation of pyruvate kinase (PK) which will allow for an increase in the flux
through gluconeogenesis.
9. PK is also allosterically inhibited by ATP and alanine. The former signals
adequate energy and the latter that sufficient substrates for gluconeogenesis are
available.
10. Conversely, a reduction in energy levels Æincreasing [ADP] lead to inhibition of
both PC and PEPCK.
11. Allosteric activation of PC occurs through acetyl-CoA

The pentose phosphate pathway

1. utilizes the 6 carbons of glucose to generate 5 carbon sugars
2. oxidize glucose and under certain conditions can completely oxidize glucose to
CO2 and water
3. generate reducing equivalents, in the form of NADPH, for reductive biosynthesis
reactions within cells
4. To provide the cell with ribose-5-phosphate (R5P) for the synthesis of the
nucleotides and nucleic acids
5. metabolize dietary pentose sugars derived from the digestion of nucleic acids
6. rearrange the carbon skeletons of dietary carbohydrates int
glycolytic/gluconeogenic intermediates
7. Enzymes that function primarily in the reductive direction utilize the
NADP+/NADPH cofactor pair as co-factors as opposed to oxidative enzymes that
utilize the NAD+/NADH cofactor pair
8. 30% of the oxidation of glucose in the liver occurs via the PPP
9. erythrocytes utilize the reactions of the PPP to generate large amounts of NADPH
used in the reduction of glutathione
10. The conversion of ribonucleotides to deoxyribonucleotides (through the action of
ribonucleotide reductase) requires NADPH as the electron source

Metabolic Disorders Associated with the Pentose Phosphate Pathway

1. Oxidative stress within cells is controlled glutathione, GSH
2. Glutathione plays the role in reducing oxidized thiols in other proteins
3. Glutathione can reduce disulfides nonenzymatically.
 4. Oxidative stress also generates peroxides that in turn can be reduced by
glutathione to generate water and an alcohol
5. Regeneration of reduced glutathione by glutathione reductase which requires the
co-factor NADPH
6. disruption in [NADPH] have a profound effect upon a cells ability to deal with
oxidative stress
Erythrocytes and the Pentose Phosphate Pathway
1. carbohydrate metabolism in red blood cells: glycolysis, the PPP and
2,3-bisphosphoglycerate (2,3-BPG) metabolism
2. Glycolysis provides ATP for membrane ion pumps and NADH for re-oxidation of
methemoglobin
3. The PPP supplies the RBC with NADPH to maintain the reduced state of
glutathione.
4. low [reduced glutathione] in RBCs Æincreased accumulation of peroxides
(H2O2) Æ weakening of the cell wall and concomitant hemolysis
5. The PPP in erythrocytes is the only pathway for these cells to produce NADPH.
Any defect in the production of NADPH could, therefore, have profound effects
on erythrocyte survival.
6. deficiencies in G6P dehydrogenase associated with resistance to the malarial
parasite; resistance from weakening of the red cell membrane (the erythrocyte is
the host cell for the parasite) and cannot sustain the parasitic life cycle long
enough for productive growth
Lipids
‧ Biological Functions of Lipids
1. Chemical structure and physical properties
Even number of C
Cis form
Trans form of FA – from dairy products and meat, such French fries,
doughnuts, and cookies
↑ LDL (bad cholesterol)
↓ HDL (good cholesterol
2. Fats and oils --- stored forms of energy in many organisms
Adipocyte and germinating seed
Contain lipase
Catalyze the hydrolysis of stored triacylglycerol
Release fatty acid for fuel
3. Advantage of stored triacylglycerol than polysaccharide
Carbon of fatty acids are more reduced than those of sugar
Oxidation of FA yields more than twice as much energy
Triacylglycerols are hydrophobic and unhydrated
Do not need to carry extra weight of water
4. Function of triacylglycerol
Energy storage
Heat insulation
Low density --- allow animal to match the buoyancy of their body to that of
their surroundings during deep dives in cold water

‧ Phospholipids and sterol

Major structure elements of biological membrane
1. The central architectural feature of biological membranes is lipid bilayer
2. Barrier to the passage of polar molecules and ions
3. Membrane-forming lipids
a. Glycerol backbond
Glycerophospholipid (Glycero-phospho-lipid)
Galactolipid (sulfolipids) chloroplast
Archaebaterial ether lipids-- typical Membrane lipid of archaebaxteria
Live in high T, low pH, high ionic strength
Have membrane lipid containing long-chain (32C) branched
hydrocarbon
By ether bond– most stable to hydrolysis at low pH and high
 temperature than ester bonds
b. Sphingosine backbond
Sphingolipid --- neuron dominant
Sphingolipids at cell surfaces are sites of biological recognition
Especially prominent in the plasma membrane of neuron
Play recognition sites on the cell surface
Few sphingolipids have been shown to correlated with special function
Carbohydrate moieties of certain sphingolipid – defined the human
blood groups
Gangliosides – present points for extracellular molecules or surface for
recognition such as embryonic development, tumor formation, cell
differentiation …..
4. Other lipids
In relatively small quantities, play crucial roles as enzyme cofactors,
electron carriers, light absorbing pigments, hydrophobic anchors for
the protein, hormone, chaperone, .. etc
a. Cholesterol
Play important role in steroid hormone production
Play role in membrane structure

Lipids as Signals
b. Eicosanoids
Including : Prostaglandin, Thromboxane, Leukotriene
Origin from C20 polyunsaturated fatty acid, the eicosaenoic acids,
particularly arachidonic acid, all-cis-5,8,11,14-eicosatetraenoic acid
Paracrine hormone-- act only on cells near the point of hormone
Locally action hormine
Low level in tissue
Rapid metabolic turnover (short-lives)
Involve in reproductive function, inflammation, fever, pain associated
with injury or disease,
Blood clot and blood pressure, gastric acid secretion

Prostaglandins (PG)
Mid-1930 Ulf Euler Sweden
1950 ne Bergstrom & Bengt Samuelsson
structural determination – five-carbon ring
PGE (ether-soluble), PGE1, PGE2,…
 PGF (phosphate, fosfat in Swedish, buffer-sluble)
mid-1960 Sweden Netherlands Biosynthesis
Function
Stimulate muscle contraction or relaxation
Affected the blood pressure to specific organ
Widely distributedin animal tissue
Regulate synthesis of cAMP – mediate diverse hormone – protaglandin
affect a wide range of cellular and tissue function
Contraction of the smooth muscle of the uterus during menstruction and
labor (PGF2a)
Wake sleep cycle (PGE2 and PGD2)
Response to epinephrine and glucagon
Elevate body temperature, cause inflammation and pain
Thromboxanes
Structural determination – five-carbon ring
Produced by platelet (also called thrombocytes)
Function
Act in the formation of blood clot
NSAIDs (Non-Steroidal Antiinflammatory Drugs) : John Vane
Aspirin, ibuprofen, and meclofenamate --- inhibit p rostaglandin H2
synthase (cyclooxygenase, COX)
Leukotriene
Produced by leukocytes
Function
Leukotriene A4 --- Leukotriene D4 --- induce contraction of the muscle
lining the airway to the lung
Overproduction of leukotrienes --- cause asthmatic attacks
Inhibition of leukotriene production – target of antiasthmatic drug,
prednisone
Biological Membranes and Transport

The Composition and Architecture of Membranes

1. Good fences make good neighbors --- Robert Frost, 1914
2. Membranes are flexible, self-sealing, and selectively permeable to polar solutes
3. Biological membrane
Flexibility --- shape changes that accompany cell growth and movement
Break and seal ---
Exocytosis : two membrane can fuse single membrane enclosed compartment
Endocytosis or cell division : can undergo fission to yield two compartment –
Selective permeability --- remain certain compounds and ions within cells and
specific cellular compartment, while exclude others
4. Cell surface
Transporter --- move specific organic solutes and inorganic ions across the
membrane
Receptor --- sense extracellular signals and trigger molecular changes in the cell
Adhesion molecules --- hold neighboring cells togethers
5. Within the cell
Cellular Process --- synthesis of lipids and certain proteins
Energy Transduction --- mitochondria and chloroplast
6. Molecular component of membrane
Protein --- peripheral protein and intergral protein
Peripheral protein ---
Associate with membrane by electrostatic interactions and H-bond with the
hydrophilic domains of integral protein
Serve as membrane-bound enzyme regulator
Limiting the mobility of integral protein
Integral protein ---
Integral protein are held in the membrane by hydrophobic Interaction with lipid and
hydrophobic domain of proteins
Associate with membrane
Release by detergents, organic solvents, or denaturants, or relative mild treatment,
such as carbonate at high pH
Types of integral protein :
Transmembrane domain
One TM
Multiple TM
hydrophobic aa sequence
 α-helix conformation
Estimate the tertiary structure of membrane protein
X-ray crystallization
Polar Lipids --- glycerophospholipid, sphingolipid, and cholesterol
Carbohydrate --- glycoprotein and glycolipid
7. Membrane fluid mosaic model --- (30 Å, ~ 20-25 aa, 1.5 Å per aa/a helix)
8. Asymmetric distribution of phospholipids between Inner and outer membrane
Changes in the asymmetric distribution have biological consequences
Platelet : blood clot
PS : outer leaflet to inner leaflet
program cell death : PS exposure on the outer surface
9. Topology of an integral membrane protein can be predicted from its primary amino
acid sequence
Hydropathy index --- ∆ G of aa move from hydrophobic solvent to water
Covalently attached lipids anchor some membrane proteins
Some membrane proteins contain one or more covalently linked lipids to provide a
hydrophobic anchor and hold the protein at the membrane surface
Long chain fatty acids
Isoprenoids
Sterols
Glycosylated PI

Membrane Dynamics
1. Effect of Heat on Membrane Structure
Gel phase
Liquid-order state
Liquid-disorder state
2. Transbilayer movement of lipids requires catalysis
3. Lipids and proteins diffuse laterally in the bilayer
4. FRAP, rate of fluorescence after photobleaching
5. Lipd raft --- sphingolipids and cholesterol cluster together in membrane rafts
6. Caveolins
Integral membrane protein with two globular domains connected by a
hairpin-shaped hydrophobic domain
Bind the protein to the cytoplasmic leaflet of the plasma membrane
Bind cholesterol in the membrane
Presence of caveolin forces the associated lipid bilayer to form caveolae (little
caves) in the surface of the cell
 Function of caveolins
membrane tracking within cells
transduction of external signals into cellular response
Appear to be located in rafts and perhaps in caveolae involved signal
transduction signaling, such as insulin receptor, GTP-binding protein,
protein kinase associated with transduction signaling, and other growth
factors.
7. Certain integral protein mediate cell-cell interaction and adhesion – integrin,
NCAM, caherin, and selectin
Integrin
αβ heterodimer
‧ Receptor –signal transducer
‧ Specific binding to extracellular ligands, such as collagen and fibronectin
‧ Determined binding sequence of integral : Arg-Gly-Asp (RGD)
‧ Integrin regulate: Platelet aggregation

Solute Transport across Membranes

Solute transport across membranes
1. Passive transport
Simple diffusion (high conc. to low conc.)
a. chemical concentration gradient
b. membrane potential gradient
2. Facilitated transport –accelerated diffusion
a. pore-facilitated transport (ion channel) --- GLUT1 (Type III)
12 TM (uniport ), Mr. 45,000, tansport glucose from blood (5mM) into
erythrocyte -- Model of glucose transport into erythrocyte by GLUT1
b. carrier-facilitated transport (ionophore)
Chloride-bicarbonate exchanger of the erythrocyte membrane
c. Three general classes of transport system --- uniport, symport (co-transport),
and antiport (co-transport)
3. Active Transport --- ( need ATP, low conc. to high conc.)
a. primary active transport --- Na+-K+ ATPase
b. secondary active transport (co-transport)
i. Symport
ii. Antiport
Biosignaling
Biosignaling --- the ability of cells to receive and act on signals from beyond the
plasma is fundamental to life

Molecular Mechanisms of Signal Transduction

1. Biosignaling
Bacterial cell --- receive constant input from membrane proteins (receptor) : toward
food, away from toxic substances
Multicellular organisms --- different function with different signals
Plants --- response to growth hormone and to variations in sunlight
Animals --- exchange information about the concentrations of ions and glucose in
extracellular fluids, interdependent metabolic activity in different
tissue, and correct signaling in embryo development
2. Molecular mechanisms of signal transduction
a. Specificity
b. Amplification
c. Desensitization / Adaptation
d. Integration
3. Ligand-receptor interaction and Scatchard Plot analysis
4. Six basic biosignaling mechanisms
a. Gated ion channel
b. Receptor enzyme
c. Serpetine receptor (G-protein coupled receptor)
d. Receptor with no intrinsic enzyme activity
e. Adhesion receptor
f. Steroid receptor

Gated Ion Channels

1. Ion channels underline electrical signaling in excitable cells
‧ The ion channel is “gated”, depend on whether the associated receptor has
been activated by binding of its specific ligand (neurotransmitter) or change its
membrane potential
‧ Na+ -K+ ATPase --- polarize the membrane (Vm= -60 to -70) : more negative
charge inside
‧ Ion flux --- redistribution of charge on the two sides of the membrane (Vm)
‧ Depolarize – efflux Cl- or influx Na+
‧ Hyperpolarize – efflux K+ to make the membrane potential more negative
2. Ion concentration in cell and extracellular fluid
a. Nicotinic acetylcholine receptor is a ligand-gated ion channel
Acetylcholine receptor (AR)
‧ AR : Found in postsynaptic membrane of neuron and in muscle fibers
(myocyte) at neuromuscular junction
‧ Acetylcholine + AR ---- trigger electrical excitation ( depolarization) of the
receiving cells
‧ AR is an allostersteric protein with two high-affinity binding sites for
acetylcholine
3. Positively cooperative
‧ Pass Na+ and Ca2+ (inward) to depolarize the plasma membrane in
postsynaptic neuron, depolarization initiate an action potential, at
neuromuscular junction, depolarization of the muscle fiber trigger muscle
contraction
4. Voltage-gated ion channel produce neuronal action potential
‧ Votage-gated Na+ channel
‧ Votage-gated K+ channel
‧ Votage-gated Ca2+ channel
‧ After open Na+ and K+ channel , open Ca2+ channel, rise in [Ca2+]i then
triggers release of acetylcholine by exocytosis into the synaptic cleft
‧ Binding to acetylcholine receptor and trigger depolarization
5. Voltage-gated ion channel convey sinalings
‧ First messenger : hormone or neurotransmitter
‧ By change the cytosolic ion concentration , ex Ca2+, to serve as the secondary
messenger
‧ By change membrane potential (Vm) and affecting other membrane proteins
that are sensitive to Vm

Receptor Enzymes
1. Have a ligand-binding domain on the extracellular surface of the plasma
membrane
2. Have an enzyme active site on the cytosolc side
3. Both two were connected by a transmembrane domain
4. Receptor enzyme is a protein kinase that phosphorylated Tyr residues in
specific target proteins
5. In plants, protein kinase – Ser / Thr phosphorylation
6. Produce secondary messenger, cGMP, cAMP : like Guanylyl cyclase receptor
7. Function of cGMP
 ‧ Secondary messenger
‧ Carry different messenger in different tissue
‧ In kidney and intenstine – change ion transport and water retention
‧ In cardiac muscle (smooth muscle) – signal for relaxation
‧ In brain – involve in development and function
8. Types of Guanylyl Cyclase
‧ Membrane form (ANF) --- kidney
‧ Membrane form (Guanylin and Endotoxin) --- plasma membrane of intestinal
epithelium cells
‧ Soluble form (sGC activated by NO; produced by NO synthase)
cGMP-PDE (different forms in different tissue)
Viagra (Inhibitor of cGMP-PDE in blood vessels of penis)

Receptor with no Intrinsic Enzyme Activity

1. When occupied their ligand, bind a soluble protein Tyr kinase
2. EPO erythroprotein (erythrocyte formation)
EPO/EPO receptor and JAK-STAT signaling
‧ EPO, 165 aa in kidney
‧ EPO + EPOR ---
‧ Receptor dimerization
‧ Bind to the soluble protein kinase JAK (Janus kinase)
‧ Phosphorykated Tyr of the cytoplasmic domain of EPOR

G Protein-Coupled Receptors and Second Messengers

1. β-adrenergic receptor (G protein-coupled receptors, GPCR)
‧ Integral protein 7TM
‧ Serpentine receptor - snake back and forth across the plasma membrane 7
times
‧ Muscle, liver and adipocyte – fuel metabolism (glycogen and fat)
2. β-adrenergic receptor is desensitized by phosphorylation
3. β-arrestin uncouples the serpentine receptor from its G protein
β-arrestin uncouple the serpentine receptor from its G protein
‧ Also act as a scaffold protein : bring together several protein kinase that
function in a cascade.
‧ Facilitate any signaling process
‧ Ex: Raf-1, MEK1, and ERK --- Insulin signaling
4. cAMP serve as the second messenger
5. IP3 and Ca 2+ serve as the secondary messenger
6. Signals act through PLC and IP3
7. Intracellular calcium concentration is in an oscillation manner
a. Calcium and Calmodulin (CaM)
‧ Calmodulin --- Mr 17,000, an acidic protein, with 4 high-affinity
Ca2+-binding site, associate with various protein, in its Ca-bound state,
modulate their activities.
‧ Regulate intracellular calcium concentration

Multivalent Scaffold Proteins and Membrane Rafts

1. One protein finds another in a signaling pathway by reversible phosphorylation of
Ser, Thr, and Tyr , by which creates docking sites for other proteins
2. Many signaling proteins are multivalents by that they can interact with several
different proteins at the same time to form multiprotein signaling complexes
3. Autoinhibition of Src Tyr kinase
4. Autoinhibition of GSK3 (Glycogen synthase kinase 3)
5. Insulin-induced formation of supermolecular signaling complexes

Regulation of Transcription by Steroid Hormones

1. Steroid hormone analog
‧ Tamoxifen
Estrogen antagonist, compete estrogen bind to estrogen receptor
Treat breast cancer
After surgery or during chemotherapy
‧ RU486
Progesterone antagonist
Compete progesterone bind to progesterone receptor

Regulation of the Cell Cycle by Protein Kinases

1. Regulation of cell cycle
Regulation of CDKs by phosphorylation
Controlled Degradation of cyclin
Regulated of CDKs and cyclin
Inhibition of CDKs
2. CDKs regulate cell division by phosphorylating critical proteins
Phosphorylation of laminin, myosin, and retinoblastoma protein
Laminin – breakdown of the nuclear envelop during mitosis
After division, CDKs phosphorylated myosin from actin filaments and inactive the
contractile machinery
 Oncogenes, Tumor Suppressor Genes, and Programmed Cell Death
1. Retinoblastoma protein
In the presence of p21, pRb remain un-phosphorylated, bind to E2F to stop the
cell cycle
2. Oncogene
Encoded Defective EGF receptor
3. Tumor suppressor gene
4. Apoptosis
Fatty Acid Catabolism

Digestion, Mobilization and Transport of Fats

1. Fatty acid oxidation
Oxidation of long chain FA to Acetyl CoA is a central energy-yielding pathway in
many organisms and tissues
Provide > 80 % energetic need
2. Acetyl CoA
TCA cycle
Converted to ketone body (in liver) which is a water soluble fuels exported to the
brain and other tissue when glucose is not available
Biosynthesis of FA (in cytosol)
A key intermediate between fat and carbohydrate metabolism
4. Fat Digestion and absorption
Triacylglycerols come from diet, adipocyte, and de novo biosynthesis from liver
Major problem: insolubility for digestion, absorption and transport of dietrary lipids
Bile salt
5. Digestion, mobilization, and transport of fats
Action of bile salts in emulsifying fats in the intestine
Synthesized from cholesterol in liver and storged in the gallbladder
6. Lipoprotein
Responsible for the transport of triacylglycerols, phospholipids, cholesterol, and
cholesteryl esters between organs
Chylomicron, VLDL, LDL, HDL
7. Chylomicron --- a lipoprotein (Apolipoprotein : a lipid binding proteins in blood)
Generalized structure of a plasma lipoprotein is spherial shape
Function of Chylomicron :
a. Carrier
b. ApoC-II :activator of lipoprotein lipase
b. Triacylglycerol is transported via chylomicrons to peripheral tissue
heart, muscle and adipose tissue
c. Remove lipid in capillaries, yielding chylomicron remnants
d. Apoprotein included: Apo A-I, Apo A-II, Apo B-48, Apo C-I, Apo
C-II, Apo C-III
8. Good and Bad Cholesterol
LDL called bad cholesterol because of link to atherosclerosis
HDL called good cholesterol because high HDL levels counter atherosclerosis by
 transporting cholesterol back to the liver from peripheral tissues
9. Fatty acid from adipocyte
Hormones trigger mobilization of stored triacylglycerols (from adipocyte)
Entry of glycerol into glycolytic pathway
10. Transport of fatty acid to mitochondria
Fatty acids are activated and transported into mitochondria
Fatty acid entry into mitochondria via the acyl-carnitine/ carnitine transporter

Oxidation of Fatty Acids

1. Fatty acid oxidation
Early experiments --- Knoop's evidence suggested fatty acids broken into two
carbon units in breakdown
Occurs in mitochondria
ATP dependent activation (need CoA)
2. β-oxidation in mitochondria --- four basic steps to regulate fatty acid breakdown
Saturated fatty acid -- even number and odd number fatty acid
Unsaturated fatty acid --- require two additional reactions
3. β-Oxidation in peroxisome --- only generate heat, but not ATP
Peroxisomes also carry out β oxidation
Plant peroxisomes and glyoxysomes use acetyl-CoA from β Oxidation as a
biosynthetic precursor
4. α-Oxidation in mitochondria
5. ω-Oxidation in ER --- Occurs in the Endoplasmic Reticulum

Ketone Bodies
1. Ketone bodies – Acetyl CoA, acetoacetate, and β – Hydroxybutyrate
2. Ketone body formation and export from the Liver
3. Acetoacetate and D- β – Hydroxybutyrate act as fuel
Lipid Biosynthesis

Biosynthesis of Fatty Acids and Ecosanoids

1. Lipid function --- principle form of stored energy in most organisms and major
constituents of cellular membranes
2. Specialized lipid serve as pigment (retinal, carotene), cofactor (Vitamin K),
detergent (bile salts), transporters (dolochols), hormone (sex hormone),
extracellular and intracellular messengers (eicosanoids, PI), anchors for membrane
proteins
3. Lipid biosynthesis
From water soluble precursors (acetate) to water insoluble product (fatty acid)
Endergonic and reductive : ATP as a source of metabolic energy and a reduced
electron carrier (NADPH)

Biosynthesis of fatty acids and eicosanoids

1. Malonyl CoA
Intermediate involved in fatty acid biosynthesis
Not involved in fatty acid breakdown
2. Malonyl-CoA is formed from acetyl-CoA and bicarbonate
ATP dependent pathway
Biotin involved – serve as a temporary carrier of CO2
3. Acetyl-CoA carboxylase (ACC)
4. Fatty acid synthesis proceeds in a repeating reaction sequence
5. Fatty acid synthesis occurs in the cytosol of many organisms but in the chloroplast
of plants
Lipid Biosynthesis and Catabolism
1. Fatty acid biosynthesis --- Cytosol (higher eukaryote) and choloplasts (plant)
2. Fatty acid degradation (catabolism)--- mitochondria (NADH)
Due to responding segregation of the electron-carrying cofactors which were used
in anabolism and catabolism
‧ [ NADH]/[NAD+] ~= very high in mitochondria
‧ Favor lipid metabolism
3. Fatty acid biosynthesis (anabolism)--- cytosol (NADPH) : favor lipid biosynthesis
In hepatocyte : [NADPH]/[NADP+] ~= 75 in cytosol
[ NADH]/[NAD+] ~= 8 X 10-4 in cytosol
Source of acetyl CoA for fatty acid biosynthesis
1. Glycolysis --- Pyruvate oxidation (major)
2.Catabolism of C-skeleton of amino acid (major)
3. β-oxidation of fatty acid (minor)
Mitochondrial inner membrane is impermeable to acetyl-CoA
Need an indirect shuttle transfer system
Citrate transporter – citrate synthase and citrate lyase
Regulation of fatty acid synthesis
1. Long-chain saturated fatty acids are synthesized from palmitate
2. Fatty acid elongation system --- In smooth ER and mitochondria
The most active elongation system of ER is C16 (palmitoyl-CoA) to C18
(Stearoyl-CoA)
Desaturation of Fatty Acids (in ER) Requires a Mix-Function Oxidase
Eicosanoids Are Formed from 20-Carbon polyunsaturated Fatty Acids
Biosynthetic routes to the major prostaglandins and thromboxane A2 (in ER)
1. The “ cyclic “ pathway from arachidonate to prostaglandin and thromboxanes
2. The “ Linear “ pathway from arachidonate to Leukotriene

Bosynthesis of Triacylglycerol
1. Fate of fatty acid biosynthesis
Incorporation into triacylglycerol for energy storage (plentiful food supply)
Incorporation into the phospholipid components of membrane (ex: rapid growth)
Both pathway begin at the same point : Formation of fatty acyl esters of glycerol
2. Triacylglycerols and glycerophospholipids are synthesized from the same
precursors (Fatty acyl-CoA and Glycerol 3-phosphate)
3. Triacylglycerols and glycerophospholipids are synthesized from phosphatidic acid
4. Triacylglycerol biosynthesis in animals is regulated by hormone

The Triacylglycerol Cycle

1. Balance between biosynthesis and degradation of triacylglycerols
2. Adipose tissue generates glycerol 3-phosphate
Glycerogenesis : short vision of Gluconeogenesis (from pyruvate to DHAP)
Presence of pyruvate carboxylase and PEP carboxyknase in adipose tissue
Glyceroneogenesis is regulated reciprocally in the liver and adipose tissue,
affecting lipid metabolism in opposite way
Lower rate of glyceroneogenesis in adipose tissue leads to more fatty acid release
Higher rate in the liver leads to more synthesis and export of triacylglycerols
Net result is an increase in flux through the triacylglycerols
Glyceroneogenesis and Type II Diabetes
1. High levels of free fatty acids in the blood interfere with glucose utilization in
muscle and promote the insulin resistance that leads to type 2 diabetes
2. Thiazolidinediones --- Reduce the levels offatty acids circulating in the blood
and increase sensitivity to insulin
3. Thiazolidinediones bind to and activate a nuclear hormone receptor (PPARr,
Peroxisome Proliferator Activated Receptor g)
4. --- induce the expression of PEP carboxykinase --- increase glyceroneogenesis

Biosynthesis of Membrane Phospholipids

1. Phospholipid synthesis occurs primarily on the surface of smooth ER and the
mitochondria inner membrane (in eukaryote)
2. Cells have two strategies for attaching phospholipid head groups
3. Eukaryotes synthesize anionic phospholipids from CDP-diacylglycerol
salvage pathway from PS to PE/PC in yeast
4. Pathway for PC synthesis from choline in mammals
5. Synthesis of plasmalogens and ether lipids
6. Biosynthesis of sphingolipids

Biosynthesis of Cholesterol
1. Intermediates in cholesterol Biosynthesis is important
2. Cholesterol has many alternative fates
Amino acid oxidation and the production of urea
Metabolic fates of amino groups
1. The catabolic pathways of ammonia and amino groups in vertebrates.
2. Most amino acids are metabolized in the liver.
3. Glutamate and glutamine play especially critical roles in nitrogen metabolism,
acting as a kind of general collection point for amino groups.
4. In skeletal muscle, excess amino groups are generally transferred to pyruvate to
form alanine.
Dietary protein is enzymatically degraded to amino acids
1. gastric mucosa secretes the gastrin
2. parietal cells secretes hydrochloric acid
3. chief cells of the gastric glands secretes pepsinogen
4. Gastric mucosa secretes gastrin
5. cholecystokinin stimulates secretion of several pancreatic enzymes with activity
optima at pH 7 to 8.
6. Acute pancreatitis is a disease caused by the zymogens of the proteolytic enzymes
are converted to their catalytically active forms prematurely, inside the pancreatic
cells
Pyridoxal phosphate participates in the transfer of
α-amino groups to
α-ketoglutarate
1. In transamination reactions, the α-amino group is transferred to the α-carbon atom
of α-ketoglutarate, leaving behind the corresponding α-keto acid analog of the
amino acid
2. All aminotransferases have pyridoxal phosphate (PLP) as prosthetic group which
is the coenzyme form of pyridoxine, or vitamin B6.
Glutamate releases its amino group as ammonia in the liver
In hepatocytes, glutamate is transported from the cytosol into mitochondria,
where it undergoes oxidative deamination catalyzed by glutamate dehydrogenase
Glutamine transports ammonia in the bloodstream
1. The free ammonia produced in tissues is combined with glutamate to yield
glutamine by the action of glutamine synthetase.
2. Glutaminase converts glutamine to glutamate and NH4
3. In the liver, the ammonia is disposed by urea synthesis.
4. Some of the glutamate produced in the glutaminase reaction may be further
processed in the liver by glutamate dehydrogenase.
5. In metabolic acidosis there is an increase in glutamine processing by the kidneys.
Assays for tissue damage
1. Analyses of certain enzyme activities in blood serum give valuable diagnostic
 information for a number of disease conditions.
2. Alanine aminotransferase (ALT; also called glutamate-pyruvate transaminase,
GPT) and aspartate aminotransferase (AST; also called glutamateoxaloacetate
transaminase, GOT) are important in the diagnosis of heart and liver damage
caused by heart attack, drug toxicity, or infection.
3. Creatine kinase (SCK) is the first heart enzyme to appear in the blood after a heart
attack.
4. Lactate dehydrogenase also leaks from injured or anaerobic heart muscle.
Glucose-alanine cycle
Alanine serves as a carrier of ammonia and of the carbon skeleton of pyruvate from
skeletal muscle to liver. The ammonia is excreted and the pyruvate is used to
produce glucose, which is returned to the muscle.
Ammonia is toxic to animals
1. A potential depletion of ATP in brain cells.
2. High levels of NH4 lead to increased levels of glutamine, which acts as an
osmotically active solute (osmolyte) in brain astrocytes, leading to cerebral edema
3. Depletion of neurotransmitters
Nitrogen excretion and the urea cycle
1. Most aquatic species are ammonotelic, excreting amino nitrogen as ammonia.
2. Most terrestrial animals are ureotelic, excreting amino nitrogen in the form of urea
3. Birds and reptiles are uricotelic, excreting amino nitrogen as uric acid.
4. Urea production occurs almost exclusively in the liver
5. Urea is produced from ammonia in five enzymatic steps: carbamoyl phosphate
synthetase I, ornithine
transcarbamoylase, argininosuccinate synthetase, argininosuccinase, arginase
6. All five enzymes are synthesized at higher rates in: starving animals, animals on
very-high-protein diets
7. carbamoyl phosphate synthetase I is allosterically activated by N-acetylglutamate.
8. Nitrogen-acquiring reactions in the synthesis of urea: carbamoyl phosphate
synthetase I and argininosuccinate synthetase
9. Careful administration of the aromatic acids benzoate or phenylbutyrate in the diet
can help lower the level of ammonia in the blood.
The Citric Acid and Urea Cycles Can Be Linked by“Krebs bicycle”:
aspartate-argininosuccinate shunt
Nonessential and essential amino acids for humans

Pathways of amino acid degradation

1. Amino acid catabolism normally account for only 10% to 15% of the human
body’s energy production
2. Ketogenic amino acids: the seven amino acids that are degraded entirely or in
part to acetoacetyl-CoA and/or acetyl-CoA—phenylalanine, tyrosine, isoleucine,
leucine, tryptophan, threonine, and lysine—can yield ketone bodies in the liver:
ketogenic amino acids
3. Glucogenic amino acids: the amino acids that are degraded to pyruvate,
α-ketoglutarate, succinyl-CoA, fumarate, and/or oxaloacetate can be converted
to glucose and glycogen by pathways
4. Both ketogenic and glucogenic amino acid: tryptophan, phenylalanine, tyrosine,
threonine, and isoleucine
Several enzyme cofactors play important roles in amino acid catabolism
1. Tetrahydrofolate (H4 folate): synthesized in bacteria, consists of substituted
pterin (6-methylpterin), p-aminobenzoate, and glutamate moieties. Convertion of
folate to tetrahydrofolate by the enzyme dihydrofolate reductase. The primary
source of one-carbon units for tetrahydrofolate is the carbon removed in the
conversion of serine to glycine, producing N5,N10-methylenetetrahydrofolate.
2. S-Adenosylmethionine (adoMet)
B12–dependent reactions in mammals: methionine synthase and L-methylmalonyl
CoA mutase
3. Biotin
4. Tetrahydrobiopterin
Amino acid Catabolism
1. phenylketonuria (PKU): phenylalanine hydroxylase, dihydrobiopterin reductase
2. Phenylacetate imparts a characteristic odor to the urine, which nurses have
traditionally used to detect PKU in infants.
3. Phenylalanine and phenylpyruvate accumulate in the blood and tissues and are
excreted in the urine—hence the name “phenylketonuria.”
4. alkaptonuria: homogentisate dioxygenase
5. albinism: tyrosine 3-monooxygenase (tyrosinase)
6. Glycine is converted to serine by serine hydroxymethyl transferase, requires the
coenzymes tetrahydrofolate and pyridoxal phosphate.
7. Tryptophan is the precursor of serotonin, melatonin and niacin
8. cysteine is synthesized from methionine and serine
Branched-Chain Amino Acids Are Not Degraded in the Liver
1. three amino acids with branched side chains (leucine, isoleucine, and valine) are
oxidized as fuels primarily in muscle, adipose, kidney, and brain tissue. These
extrahepatic tissues contain an aminotransferase, absent in liver.
2. branched-chain α-keto acid dehydrogenase complex needs Five cofactors
(thiamine pyrophosphate, FAD, NAD, lipoate, and coenzyme A)
3. This enzyme is defective in people with maple syrup urine disease.
Biosynthesis of Amino Acids, Nucleotides, and Related Molecules
Overview of Nitrogen Metabolism
1. The soil bacteria use NO3- as the ultimate electron acceptor and generates a
transmembrane proton gradient to synthesize ATP.
2. Ammonia is incorporated into biomolecules through glutamate and glutamine:
glutamate dehydrogenase and glutamine synthetase
3. Glutamine synthetase is a primary regulatory point in nitrogen metabolism
4. Pyridoxal phosphate is required for transamination reactions involving glutamate
and for other amino acid transformations.
5. One-carbon transfers require S-adenosylmethionine and tetrahydrofolate.
Biosynthesis of Amino Acids
1. All amino acids are derived from intermediates in glycolysis, the citric acid
cycle, or the pentose phosphate pathway
2. Nitrogen enters these pathways by way of glutamate and glutamine.
3. glutamate is formed by reductive amination of α-ketoglutarate and serves as
the precursor of glutamine, proline, and arginine.
4. Alanine and aspartate (and thus asparagine) are formed from pyruvate and
oxaloacetate, respectively, by transamination.
5. α-Ketoglutarate gives rise to glutamate, glutamine, proline, and arginine
6. Serine, glycine, and cysteine are derived from 3-phosphoglycerate
7. Serine (three carbons) is the precursor of glycine (two carbons) through
removal of a carbon atom by serine hydroxymethyltransferase.
Tetrahydrofolate accepts the β carbon (C-3) of serine, which forms a
methylene bridge between N-5 and N-10 to yield
N5,N10-methylenetetrahydrofolate, and needs PLP as a cofactor.
8. Biosynthesis of cysteine from homocysteine and serine in mammals.
9. Chorismate is a key intermediate in the synthesis of tryptophan, phenylalanine,
and tyrosine
10. Histidine biosynthesis uses precursors of purine biosynthesis
11. The amino acid biosynthetic pathways are subject to allosteric end-product
inhibition; the regulatory enzyme is usually the first in the sequence.
Regulation of the various synthetic pathways is coordinated.
Glycine is a precursor of porphyrins
1. Porphyrins are constructed from porphobilinogen
2. protoporphyrin
3. The iron atom is incorporated into protoporphyrin by ferrochelatase.
4. Genetic defects in the biosynthesis of porphyrins can lead to the accumulation
of pathway intermediates, causing a variety of human diseases known
 collectively as porphyries
5. Heme is the source of bile pigments
6. Heme oxygenase converts heme to biliverdin, free Fe2+ and and CO.
7. The Fe2+ is quickly bound by ferritin.
8. Biliverdin is converted to bilirubin by biliverdin reductase.
9. When you are bruised, the black and/or purple color results from hemoglobin
released from damaged erythrocytes. Over time, the color changes to the green
of biliverdin, and then to the yellow of bilirubin.
10. Bilirubin is largely insoluble, and it travels in the bloodstream as a complex
with serum albumin.
11. In the liver, bilirubin is transformed to the bile pigment bilirubin diglucuronide.
This product is secreted into the small intestine, where microbial enzymes
convert it to urobilinogen. Some urobilinogen is reabsorbed into the blood and
transported to the kidney, where it is converted to urobilin, the compound that
gives urine its yellow color.
12. Urobilinogen remaining in the intestine is converted to stercobilin which
imparts the red-brown color to feces.
13. Impaired liver function or blocked bile secretion auses bilirubin to leak from
the liver into the blood, resulting in jaundice.
14. Newborn infants develop jaundice because they have not yet produced enough
glucuronyl bilirubin transferase to process their bilirubin.
15. The CO at the very low concentrations generated during heme degradation it
appears to have some regulatory and/or signaling functions. It acts as a
vasodilator and have some regulatory effects on neurotransmission.
16. Bilirubin is the most abundant antioxidant in mammalian tissues and is
responsible for most of the antioxidant activity in serum.
Amino Acids Are Precursors of Creatine and Glutathione
1. Phosphocreatine is an important energy buffer in skeletal muscle.
2. Creatine is synthesized from glycine, arginine and S-adenosylmethionine
3. Glutathione (GSH) can be thought of as a redox buffer. It is derived from glycine,
glutamate, and cysteine
4. Glutathione helps maintain the sulfhydryl groups of proteins in the reduced state
and the iron of heme in the ferrous state, and it serves as a reducing agent for
glutaredoxin in deoxyribonucleotide synthesis.
5. Glutathione peroxidase contains a covalently bound selenium (Se) atom in the
form of selenocysteine which is essential for its activity.
6. D-amino acids serve some special functions in the structure of bacterial cell
walls and peptide antibiotics.
Biological Amines Are Products of Amino Acid Decarboxylation
1. Tyrosine gives rise to a family of catecholamines that includes dopamine,
norepinephrine, and epinephrine.
2. The neurological disorder Parkinson’s disease is associated with an
underproduction of dopamine, and it has traditionally been treated by
administering L-dopa.
3. Overproduction of dopamine in the brain may be linked to psychological
disorders such as schizophrenia.
4. Glutamate decarboxylation gives rise to γ-aminobutyrate (GABA), an inhibitory
neurotransmitter. Its underproduction is associated with epileptic seizures.
5. Serotonin, is derived from tryptophan
6. Histidine undergoes decarboxylation to histamine, a powerful vasodilator in
animal tissues. Histamine is released in large amounts as part of the allergic
response, and it also stimulates acid secretion in the stomach.
7. Histamine receptor antagonist cimetidine (Tagamet), a structural analog of
histamine: promotes the healing of duodenal ulcers by inhibiting secretion of
gastric acid.
8. Polyamines such as spermine and spermidine, involved in DNA packaging, are
derived from methionine and ornithine. The first step is decarboxylation of
ornithine, a precursor of arginine. Ornithine decarboxylase, a PLP-requiring
enzyme, is the target of several powerful inhibitors used as pharmaceutical
agents.
Arginine Is the Precursor for Biological Synthesis of Nitric Oxide
1. In humans NO plays a role including neurotransmission, blood clotting, and the
control of blood pressure.
2. Nitric oxide is synthesized from arginine in an NADPH-dependent reaction
catalyzed by nitric oxide synthase.
3. NO synthesis is stimulated by interaction of nitric oxide synthase with
Ca2+-calmodulin.
Biosynthesis and Degradation of Nucleotides
1. Nucleotides are the precursors of DNA and RNA. They are essential carriers of
chemical energy- ATP and GTP. They are components of the cofactors NAD,
FAD, S-adenosylmethionine, and coenzyme A, as well as of activated
biosynthetic intermediates such as UDP-glucose and CDP-diacylglycerol.
2. Two types of pathways lead to nucleotides: the de novo pathways and the
salvage pathways. De novo synthesis of nucleotides begins with their
metabolic precursors: amino acids, ribose 5-phosphate, CO2, and NH3. Salvage
pathways recycle the free bases and nucleosides released from nucleic acid
 breakdown.
3. The free bases guanine, adenine, thymine, cytidine, and uracil are not
intermediates in de novo pathways.
4. The purine ring structure is built up one or a few atoms at a time, attached to
ribose throughout the process. The pyrimidine ring is synthesized as orotate,
attached to ribose phosphate, and then converted to the common pyrimidine
nucleotides required in nucleic acid synthesis.
5. Although the free bases are not intermediates in the de novo pathways, they are
intermediates in some of the salvage pathways.
6. Several important precursors are shared by the de novo pathways for synthesis
of pyrimidines and purines. Phosphoribosyl pyrophosphate (PRPP) is
important in both, and in these pathways the structure of ribose is retained in
the product nucleotide.
7. In the de novo purine pathway,the enzymes are present as large, multienzyme
complexes in the cell.
8. The cellular pools of nucleotides (other than ATP) are quite small, perhaps 1%
or less of the amounts required to synthesize the cell’s DNA. Therefore, cells
must continue to synthesize nucleotides during nucleic acid synthesis.
9. The origin of the carbon and nitrogen atoms of the purine ring system: the
amide N of glutamine contributes N3 and N9; aspartate is the N1 donor;
glycine contributes C4, C5 and N7; formate is the donor of C2 and C8 and
supplied in the form of N10-formyltetrahydrofolate; CO2 contributes the C6.
10. The first intermediate with a complete purine ring is inosinate (IMP).
11. Purine nucleotide biosynthesis is regulated by feedback inhibition:
glutamine-PRPP amidotransferase, IMP dehydrogenase, and ribose phosphate
pyrophosphokinase.
12. Pyrimidine nucleotides are made from aspartate, PRPP, and carbamoyl
phosphate.
13. De novo pyrimidine nucleotide biosynthesis proceeds in a somewhat different
manner from purine nucleotide synthesis; the six-membered pyrimidine ring is
made first and then attached to ribose 5-phosphate.
14. In animals the carbamoyl phosphate required in urea synthesis is made in
mitochondria by carbamoyl phosphate synthetase I, whereas the carbamoyl
phosphate required in pyrimidine biosynthesis is made in the cytosol by a
different form of the enzyme, carbamoyl phosphate synthetase II.
15. Six enzymes are needed in pyrimidine nycleotide synthesis: carbamoyl
phosphate synthetase II, aspartate transcarbamoylase, dihydroorotase,
dihydroorotate dehydrogenase, orotate phosphoribosyltransferase and
 orotidylate decarboxylase, only dihydroorotate dehydrogenase is a
mitochondrial enzyme.
16. Allosteric regulation of aspartate transcarbamoylase by CTP (negative) and
ATP (positive).
Ribonucleotides Are the Precursors of Deoxyribonucleotides
1. Reduction of ribonucleotides to deoxyribonucleotides by ribonucleotide
reductase.
2. The reaction: conversion of NDP to dNDP is catalyzed by ribonucleotide
reductase.
3. Electrons are transmitted to the enzyme from NADPH by (a) glutaredoxin or (b)
thioredoxin.
4. The sulfide groups in glutaredoxin reductase are contributed by two molecules of
bound glutathione
5. Thioredoxin reductase is a flavoenzyme, with FAD as prosthetic group.
Thymidylate Is Derived from dCDP and dUMP
1. The immediate precursor of thymidylate (dTMP) is dUMP.
2. The dUTP is converted to dUMP by a dUTPase. The latter reaction must be
efficient to keep dUTP pools low and prevent incorporation of uridylate into
DNA.
3. Conversion of dUMP to dTMP by thymidylate synthase and dihydrofolate
reductase. Serine hydroxymethyltransferase is required for regeneration of the
N5,N10-methylene form of tetrahydrofolate.
Degradation of Purines and Pyrimidines Produces Uric Acid and Urea,
Respectively
1. Purine nucleotides are degraded by 5’-nucleotidase’, adenosine deaminase, and
xanthine oxidase.
2. Xanthine oxidase is a flavoenzyme with an atom of molybdenum and four
iron-sulfur centers in its prosthetic group.
3. Uric acid is the excreted end product of purine catabolism in primates, birds, and
some other animals.
4. The pathways for degradation of pyrimidines generally lead to NH4+ production
and thus to urea synthesis.
5. Adenosine deaminase (ADA) deficiency leads to severe immunodeficiency
disease in which T lymphocytes and B lymphocytes do not develop properly.
Lack of ADA leads to a 100-fold increase in the cellular concentration of dATP,
a strong inhibitor of ribonucleotide reductase.
Purine and Pyrimidine Bases Are Recycled by Salvage Pathways
1. One of the primary salvage pathways consists of a single reaction catalyzed by
 adenosine phosphoribosyltransferase, in which free adenine reacts with PRPP to
yield the corresponding adenine nucleotide
2. Free guanine and hypoxanthine are salvaged in the same way by
hypoxanthine-guanine phosphoribosyltransferase.
3. A genetic lack of hypoxanthine-guanine phosphoribosyltransferase activity, seen
almost exclusively in male children, results in Lesch-Nyhan syndrome. Children
with this genetic disorder are sometimes poorly coordinated and mentally
retarded. In addition, they are extremely hostile and show compulsive
self-destructive tendencies: they mutilate themselves by biting off their fingers,
toes, and lips.
Excess Uric Acid Causes Gout
1. Gout occurs predominantly in males and often involves an underexcretion of
urate.
2. Gout is effectively treated by a combination of nutritional and drug therapies.
Foods especially rich in nucleotides and nucleic acids are withheld from the diet.
Major alleviation of the symptoms is provided by the drug allopurinol which
inhibits xanthine oxidase
3. Allopurinol is a substrate of xanthine oxidase, which converts allopurinol to
oxypurinol (alloxanthine). Oxypurinol inactivates the reduced form of the
enzyme by remaining tightly bound in its active site. When xanthine oxidase is
inhibited, the excreted products of purine metabolism are xanthine and
hypoxanthine, which are more water soluble than uric acid and less likely to
form crystalline deposits.
Pathways for Degradation of Pyrimidine
1. The pathways for degradation of pyrimidines generally lead to NH4+ production
and thus to urea synthesis.
2. Thymine is degraded to methylmalonylsemialdehyde and further degraded
through propionyl-CoA and methylmalonyl-CoA to succinyl-CoA.
Many Chemotherapeutic Agents Target Enzymes in the Nucleotide Biosynthetic
Pathways
1. glutamine amidotransferase: azaserine and acivicin
2. thymidylate synthase: fluorouracil
3. dihydrofolate reductase: methotrexate, aminopterin
4. bacterial dihydrofolate reductase: trimethoprim
Hormonal regulation and integration of mammalian metabolism
Hormones: diverse structures for diverse functions
1. In neuronal signaling, the chemical messenger (neurotransmitter; acetylcholine,
for example) may travel only a fraction of a micrometer, across the synaptic cleft
to the next neuron in a network. In hormonal signaling, the
messengers—hormones—are carried in the bloodstream to neighboring cells or
to distant organs and tissues; they may travel a meter or more before
encountering their target cell.
2. Epinephrine and norepinephrine serve as neurotransmitters in certain synapses of
the brain and smooth muscle and as hormones that regulate fuel metabolism in
liver and muscle.
3. The structure of thyrotropin-releasing hormone (TRH): a derivative of the
tripeptide Glu–His–Pro.
Hormones Act through Specific High-Affinity Cellular Receptors
1. hormone-receptor interactions can be quantified by Scatchard analysis which
yields a quantitative measure of affinity and the number of hormone-binding
sites in a preparation of receptor.
2. The intracellular consequences of hormone-receptor interaction are of at least six
general types: (1) a change in membrane potential results from the opening or
closing of a hormone-gated ion channel; (2) a receptor enzyme is activated by the
extracellular hormone; (3) a second messenger (such as cAMP or inositol
trisphosphate) generated inside the cell acts as an allosteric regulator of one or
more enzymes; (4) a receptor with no intrinsic enzyme activity recruits a soluble
protein kinase in the cytosol, which passes on the signal; (5) an adhesion receptor
on the cell surface interacts with molecules in the extracellular matrix and
conveys information to the cytoskeleton; or (6) a steroid or steroidlike molecule
causes a change in the level of expression (transcription of DNA into mRNA) of
one or more genes, mediated by a nuclear hormone receptor protein.
3. Water-soluble peptide and amine hormones (insulin and epinephrine, for
example) act extracellularly by binding to cell surface receptors that span the
plasma membrane. When the hormone binds to its extracellular domain, the
receptor undergoes a conformational change analogous to that produced in an
allosteric enzyme by binding of an effector molecule. The conformational change
triggers the downstream effects of the hormone.
4. Water-insoluble hormones (steroid, retinoid, and thyroid hormones) readily pass
through the plasma membrane of their target cells to reach their receptor proteins
in the nucleus. With this class of hormones, the hormone-receptor complex itself
carries the message; it interacts with DNA to alter the expression of specific
 genes, changing the enzyme complement of the cell and thereby changing
cellular metabolism.
5. Hormones that act through plasma membrane receptors generally trigger very
rapid physiological or biochemical responses.
6. The thyroid hormones and the sex (steroid) hormones promote maximal
responses in their target tissues only after hours or even day.
7. The fast-acting hormones lead to a change in the activity of one or more
preexisting enzymes in the cell, by allosteric mechanisms or covalent
modification. The slower-acting hormones generally alter gene expression,
resulting in the synthesis of more or less of the regulated protein(s).
Hormones Are Chemically Diverse
1. Peptide, amine, and eicosanoid hormones act from outside the target cell via
surface receptors. Steroid, vitamin D, retinoid, and thyroid hormones enter the
cell and act through nuclear receptors. Nitric oxide also enters the cell, but
activates a cytosolic enzyme, guanylyl cyclase.
2. Hormones can be classified by the way they get from the point of their release
to their target tissue: endocrine hormones, paracrine hormones and autocrine
hormones.
3. Peptide hormones include the pancreatic hormones insulin, glucagon, and
somatostatin, the parathyroid hormone, calcitonin, and all the hormones of the
hypothalamus and pituitary. These hormones are synthesized on ribosomes in
the form of longer precursor proteins (prohormones), then packaged into
secretory vesicles and proteolytically cleaved to form the active peptides.
4. Mature insulin is formed from its larger precursor preproinsulin by proteolytic
processing. Removal of the signal sequence at the amino terminus of
preproinsulin and formation of three disulfide bonds produces proinsulin.
Further proteolytic cuts remove the C peptide from proinsulin to produce mature
insulin, composed of A and B chains.
5. Catecholamine hormones are epinephrine (adrenaline) and norepinephrine
(noradrenaline). They are synthesized from tyrosine. Catecholamines produced
in the brain and in other neural tissues function as neurotransmitters, but
epinephrine and norepinephrine are also hormones, synthesized and secreted by
the adrenal glands.
6. The eicosanoid hormones (prostaglandins, thromboxanes, and leukotrienes)
are derived from the 20-carbon polyunsaturated fatty acid arachidonate
enzymatically released from membrane phospholipids by phospholipase A2. The
eicosanoid hormones are paracrine hormones.
7. Prostaglandins promote the contraction of smooth muscle, including that of
 the intestine and uterus (and can therefore be used medically to induce labor).
They also mediate pain and inflammation in all tissues. Many antiinflammatory
drugs act by inhibiting steps in the prostaglandin synthetic pathway.
Thromboxanes regulate platelet function and therefore blood clotting.
Leukotrienes LTC4 and LTD4 act through plasma membrane receptors to
stimulate contraction of smooth muscle in the intestine, pulmonary airways, and
trachea. They are mediators of the severe immune response called anaphylaxis.
8. The steroid hormones (adrenocortical hormones and sex hormones) are
synthesized from cholesterol.
9. Glucocorticoids (such as cortisol) primarily affect the metabolism of
carbohydrates; mineralocorticoids (such as aldosterone) regulate the
concentrations of electrolytes in the blood.
10. The syntheses of androgens (testosterone) and estrogens involves cytochrome
P-450 enzymes that cleave the side chain of cholesterol and introduce oxygen
atoms. These hormones affect sexual development, sexual behavior, and a
variety of other reproductive and nonreproductive functions.
11. All steroid hormones act through nuclear receptors to change the level of
expression of specific genes. Recent evidence indicates that they also have more
rapid effects, mediated by receptors localized in the plasma membrane.
12. Calcitriol (1,25-dihydroxycholecalciferol) is produced from vitamin D by
enzymecatalyzed hydroxylation in the liver and kidneys.
13. Vitamin D is obtained in the diet or by photolysis of 7-dehydrocholesterol in
skin exposed to sunlight.
14. Calcitriol works in concert with parathyroid hormone in Ca2+ homeostasis,
regulating [Ca2+] in the blood and the balance between Ca2+ deposition and Ca2+
mobilization from bone.
15. Acting through nuclear receptors, calcitriol activates the synthesis of an
intestinal Ca2+ binding protein essential for uptake of dietary Ca2+. Inadequate
dietary vitamin D or defects in the biosynthesis of calcitriol result in serious
diseases such as rickets, in which bones are weak and malformed.
16. Retinoids are potent hormones that regulate the growth, survival, and
differentiation of cells via nuclear retinoid receptors. The prohormone retinol is
synthesized from vitamin A, primarily in liver and many tissues convert retinol
to the hormone retinoic acid (RA).
17. All tissues are retinoid targets. In adults, the most significant targets include
cornea, skin, epithelia of the lungs and trachea, and the immune system. RA
regulates the synthesis of proteins essential for growth or differentiation.
Excessive vitamin A can cause birth defects, and pregnant women are advised
 not to use the retinoid creams that have been developed for treatment of severe
acne.
18. The thyroid hormones T4 (thyroxine) and T3 (triiodothyronine) are synthesized
from the precursor protein thyroglobulin. Two iodotyrosine residues condense
to form the precursor to thyroxine. When needed, thyroxine is released by
proteolysis. Condensation of monoiodotyrosine with diiodotyrosine produces T3,
which is also an active hormone released by proteolysis. The thyroid hormones
act through nuclear receptors to stimulate energy-yielding metabolism,
especially in liver and muscle, by increasing the expression of genes encoding
key catabolic enzymes.
19. Nitric oxide is synthesized from molecular oxygen and the guanidino nitrogen
of arginine in a reaction catalyzed by NO synthase.
20. NO acts near its point of release, entering the target cell and activating the
cytosolic enzyme guanylyl cyclase, which catalyzes the formation of the second
messenger cGMP.

Tissue-Specific Metabolism: The Division of Labor

1. Brain: Transports ions to maintain membrane potential; integrates inputs from
body and surroundings; sends signals to other organs.
(1). The neurons of the adult mammalian brain normally use only glucose as fuel.
(2). Astrocytes can oxidize fatty acids.
(3). It uses O2 at a fairly constant rate, accounting for almost 20% of the total O2
consumed by the body at rest.
(4). The brain oxidizes β-hydroxybutyrαte via acetyl-CoA becomes important
during prolonged fasting or starvation.
(5). Energy is required to create and maintain an electrical potential across the
neuronal plasma membrane.
 (6). The technique of positron emission tomography (PET) shows glucose
metabolism in the brain.
2. Pancreas: Secretes insulin and glucagon in response to changes in blood glucose
concentration.
3. Liver: Processes fats, carbohydrates, proteins from diet; synthesizes and
distributes lipids, ketone bodies, and glucose for other tissues; converts excess
nitrogen to urea.
(1). Metabolic pathways for glucose 6-phosphate in the liver.
(2). Metabolism of amino acids in the liver.
(3). Metabolism of fatty acids in the liver.
(4). Glucose-alanine cycle
(5). Fatty acids are the primary oxidative fuel in the liver.
(6). Ketone bodies may be regarded as a transport form of acetyl groups. They
can supply a significant fraction of the energy in some extrahepatic
tissues-up to onethird in the heart, and as much as 60% to 70% in the brain
during prolonged fasting.
(7). Certain nutrients are stored in the liver, including Fe ions and vitamin A.
4. Portal vein: Carries nutrients from intestine to liver.
5. Small intestine: Absorbs nutrients from the diet, moves them into blood or
lymphatic system.
6. Lymphatic system: Carries lipids from intestine to liver.
7. Adipose tissue: Synthesizes, stores, and mobilizes triacylglycerols.
(1). Adipocytes have an active glycolytic metabolism, use the citric acid cycle to
oxidize pyruvate and fatty acids, and carry out oxidative phosphorylation.
(2). The release of fatty acids from adipocytes is greatly accelerated by
epinephrine, which stimulates the cAMP-dependent phosphorylation of
perilipin; this gives triacylglycerol lipase access to triacylglycerols in the
lipid droplet. Insulin counterbalances this effect of epinephrine, decreasing
the activity of triacylglycerol lipase.
8. Skeletal muscle: Uses ATP to do mechanical work.
(1). Cori cycle
(2). The heart uses as its fuel mainly free fatty acids
(3). The heart muscle have small amounts of reserve energy in the form of
phosphocreatine,
The Pancreas Secretes Insulin or Glucagon in Response to Changes in Blood
Glucose
1. Each cell type of the islets produces a single hormone: α cells produce glucagon;
β cells, insulin; and δ cells, somatostatin.
2. Glucose regulation of insulin secretion by pancreatic β cells. When the blood
glucose level is high, active metabolism of glucose in the β cell raises
intracellular [ATP], which leads to closing of K+
channels in the plasma
membrane, depolarizing the membrane. In response to the change in membrane
potential, voltage-gated Ca2+ channels in the plasma membrane open, allowing
Ca2+ to flow into the cell; this raises the cytosolic [Ca2+] enough to trigger insulin
release by exocytosis.
3. The well-fed state: the lipogenic liver. Immediately after a calorie-rich meal,
glucose, fatty acids, and amino acids enter the liver. Insulin released in response
to the high blood glucose concentration stimulates glucose uptake by the tissues.
Some glucose is exported to the brain for its energetic needs, and some to fat and
muscle tissue. In the liver, excess glucose is oxidized to acetyl-CoA, which is
used to synthesize fatty acids for export as triacylglycerols in VLDLs to fat and
muscle tissue. The NADPH necessary for lipid synthesis is obtained by oxidation
of glucose in the pentose phosphate pathway. Excess amino acids are converted
to pyruvate and acetyl- CoA, which are also used for lipid synthesis. Dietary fats
 move via the lymphatic system, as chylomicrons, from the intestine to muscle
and fat tissues.
4. The fasting state: the glucogenic liver. After some hours without a meal, the
liver becomes the principal source of glucose for the brain. Liver glycogen is
broken down, and the glucose 1-phosphate produced is converted to glucose
6-phosphate, then to free glucose, which is released into the bloodstream. Amino
acids from the degradation of proteins and glycerol from the breakdown of TAGs
in adipose tissue are used for gluconeogenesis. The liver uses fatty acids as its
principal fuel, and excess acetyl-CoA is converted to ketone bodies for export to
other tissues for fuel; the brain is especially dependent on this fuel when glucose
is in short supply.
5. Effect of Insulin on Blood Glucose

6. Fuel metabolism in the liver during prolonged fasting or in uncontrolled

diabetes mellitus. After depletion of stored carbohydrates, proteins become an
important source of glucose, produced from glucogenic amino acids by
gluconeogenesis. Fatty acids imported from adipose tissue are converted to
ketone bodies for export to the brain.
Effect of Glucagon on Blood Glucose
7. Concentrations of fatty acids, glucose, and ketone bodies in the plasma
during the first week of starvation. The level of glucose in the blood begins to
diminish after two days of fasting. The level of ketone bodies, almost
immeasurable before the fast, rises dramatically after 2 to 4 days of fasting.
These water-soluble ketones, acetoacetate and β-hydroxybutyrate, supplement
glucose as an energy source during a long fast. Fatty acids cannot serve as a fuel
for the brain; they do not cross the blood-brain barrier.
8.

9. Cortisol Signals Stress, Including Low Blood Glucose

(1). Stressors (anxiety, fear, pain, hemorrhage, infections, low blood glucose,
starvation) stimulate release
(2). of the corticosteroid hormone cortisol from the adrenal cortex.
(3). Cortisol acts on muscle, liver, and adipose tissue to supply the organism
with fuel to withstand the stress.
(4). Cortisol alters metabolism by changing the kinds and amounts of certain
enzymes synthesized in its target cell, rather than by regulating the activity
of existing enzyme molecules.
(5). In adipose tissue, cortisol leads to an increase in the release of fatty acids
from stored TAGs. The fatty acids are exported to serve as fuel for other
tissues, and the glycerol is used for gluconeogenesis in the liver.
(6). Cortisol stimulates the breakdown of muscle proteins and the export of
amino acids to the liver, where they serve as precursors for
gluconeogenesis.
(7). In the liver, cortisol promotes gluconeogenesis by stimulating synthesis of
the key enzyme PEP carboxykinase. Glucose produced in this way is stored
in the liver as glycogen or exported immediately to tissues that need glucose
 for fuel.
(8). The effects of cortisol therefore counterbalance those of insulin.
10. In diabetes, insulin is either not produced or not recognized by the tissues, and
the uptake of blood glucose is compromised. When blood glucose levels are high,
glucose is excreted. Tissues then depend on fatty acids for fuel (producing ketone
bodies) and degrade cellular proteins to provide glucogenic amino acids for
glucose synthesis. Uncontrolled diabetes is characterized by high glucose levels
in the blood and urine and the production and excretion of ketone bodies.
11. The characteristic metabolic change in diabetes is excessive but incomplete
oxidation of fatty acids in the liver. The acetyl-CoA produced by β oxidation
cannot be completely oxidized by the citric acid cycle, because the high
[NADH]/[NAD+] ratio produced by β oxidation inhibits the cycle. Accumulation
of acetyl-CoA leads to overproduction of the ketone bodies which cannot be used
by extrahepatic tissues as fast as they are made in the liver.
Obesity and the Regulation of Body Mass
1. Adipose tissue produces leptin, a hormone that regulates feeding behavior and
energy expenditure so as to maintain adequate reserves of fat. Leptin production
and release increase with the number and size of adipocytes.
2. Leptin acts on receptors in the arcuate nucleus of the hypothalamus, causing the
release of anorexigenic peptides, including -MSH, that act in the brain to inhibit
eating. Leptin also stimulates sympathetic nervous system action on adipocytes,
leading to uncoupling of mitochondrial oxidative phosphorylation, with
consequent thermogenesis.
3. The signal-transduction mechanism for leptin involves phosphorylation of the
JAK-STAT system. On phosphorylation by JAK, STATs can bind to regulatory
regions in nuclear DNA and alter the expression of genes for the proteins that set
the level of metabolic activity and determine feeding behavior. Insulin acts on
receptors in the arcuate nucleus, with results similar to those caused by leptin.
4. The hormone adiponectin stimulates fatty acid uptake and oxidation and inhibits
fatty acid synthesis. Its actions are mediated by AMPK.
5. Ghrelin, a hormone produced in the stomach, acts on orexigenic neurons in the
arcuate nucleus to produce hunger before a meal. PYY3–36, a peptide hormone
of the intestine, acts at the same site to lessen hunger after a meal.
PROKARYOTIC CHROMOSOME STRUCTURE
The Escherichia coli chromosome
The E. coli chromosome is a closed-circular DNA of length 4.6 million base pairs,
which resides in a region of the cell called the nucleoid. In normal growth, the DNA is
being replicated continuously.

DNA domains
The genome is organized into 50-100 large loops or domains of 50-100 kb in length,
which are constrained by binding to a membrane-protein complex.

Supercoiling of the genome

The genome is negatively supercoiled. Individual domains may be topologically
independent, that is they may be able to support different levels of supercoiling.

DNA-binding proteins
The DNA domains are compacted by wrapping around nonspecific DNA-binding
proteins such as HU and H-NS (histone-like proteins). These proteins constrain about
half of the supercoiling of the DNA. Other molecules such as integration host factor,
RNA polymerase and mRNA may help to organize the nucleoid.
DNA REPLICATION: AN OVERVIEW
Semi-conservative mechanism
During replication, the strands of the double helix separate and each acts as a
template to direct the synthesis of a complementary daughter strand using
deoxyribonucleoside 5’-triphosphates as precursors. Thus, each daughter cell receives
one of the original parental strands. This mechanism can be demonstrated
experimentally by density labeling experiments.

Replicons, origins and termini

Small chromosomes, such as those of bacteria and virus, replicate as single units
called replicons. Replication begins from a unique site, the origin, and proceed,
usually bidirectionally, to the terminus. Large enukaryotic chromosomes contain
multiple replicons, each with its own origin, which fuse as they replicate. Origins tend
to be AT-rich to make opening easier.

Semi-discontinuous replication
At each replication fork, the leading strand is synthesized as one continuous piece
while the lagging strand is made discontinuously as short fragments in the reverse
direction. These frangments are 1000-2000 nt long in prokaryotes and 100-200 nt
long in eukaryotes. They are joined by DNA ligase. This mechanism exists because
DNA can be synthesized in a 5＇→3＇direction.

RNA priming
The leading strand and all lagging strand fragments are primed by synthesis of a short
piece of RNA which is then elongated with DNA. The primers are removed and
replaced by DNA before ligation. This mechanism helps to maintain high replicational
fidelity.
MUTAGENESIS
Mutation
Mutations are heritable permanent changes in the base sequence of DNA. Point
mutations may be transitions (e.g. G.．C→A．T) or transversions (e.g. G.．C→T．
A). Deletions and insertions involve the loss or addition of bases and can cause
frameshifts in reading the genetic code. Silent mutations have no phenotypic effect,
while missense and nonsense mutations change the amino acid sequence of the
encoded protein.

Replication fidelity
The high accuracy of DNA replication (one error per 1010 bases incorporated)
depends on a combination of proper base pairing of template strand and incoming
nucleotide in the active site of the DNA polymerase, proofreading of the incorporated
base by 3’ →5’ exonuclease and mismatch repair.

Physical mutagens
Ionizing (e.g. X- and γ-rays) and nonionizing (e.g. UV) radiation produce a variety
of DNA lesions. Pyrimidine dimmers are the commonest product of UV irradiation.

Chemical mutagens
Base analogs can mispair during DNA replication to cause mutations. Nitrous acid
deaminates cytosine and adenine. Alkylating and arylating agents generate a variety of
adducts that can block transcription and replication and cause mutations by direct or,
more commonly, indirect mutagenesis. Most chemical mutagens are carcinogenic.

Direct mutagenesis
If a base analog or modified base whose base pairing properties are different from
the parent base is not removed by a DNA repair mechanism before passage of a
replication fork, then an incorrect base will be incorporated. A second round of
replication fixes the mutation permanently in the DNA.

Indirect mutagenesis
Most lesions in DNA are repaired by error-free direct reversal or excision repair
mechanisms before passage of a replication fork. If this is not possible, translesion
DNA synthesis may take place and one or more incorrect bases become incorporated
opposite the lesion. Proofreading may be suppressed during this process
BASIC PRINCIPLES OF TRANSCRIPTION
Transcription: an overview
Transcription is the synthesis of a single-stranded RNA from a double-stranded
DNA template. RNA synthesis occurs in the 5＇→3’ direction and its sequence
corresponds to that of the DNA strand which is known as the sense strand.

Initiation
RNA polymerase is the enzyme responsible for transcription. It binds to specific
DNA sequences called promoter to initiate RNA synthesis. These sequences are
upstream (to the 5’-end) of the region that codes for protein, and they contain short,
conserved DNA sequences which are common to different promoters. The RNA
polymerase binds to the dsDNA at a promoter sequence, resulting in local DNA
unwinding. The position of the first synthesized base of the RNA is called the start
site and is designated as position+1.

Elongation
RNA polymerase moves along the DNA and sequentially synthesizes the RNA
chain. DNA is unwond ahead of the moving polymerase, and the helix is reformed
behind it.

Termination
RNA polymerase recognizes the terminator which causes no further ribonucleotides
to be incorporated. This sequence is commonly a hairpin structure. Some terminators
require an accessory factor called rho for termination.
THE THREE RNA POLYMERASES: CHARACTERIZATION AND
FUNCTION
Eukaryotic RNA polymerases
Three eukaryotic polymerases transcribe different sets of genes. Their activities are
distinguished by their different sensitivities to the fungal toxin α-amanitin.
„ RNA polymerase I is located in the nucleoli. It is responsible for the synthesis
of the precursors of most rRNAs.
„ RNA polymerase II is located in the nucleoplasm and is responsible for the
synthesis of mRNA precursors and some small nuclear RNAs.
„ RNA polymerase III is located in the nucleoplasm. It is responsible for the
synthesis of the precursors of 5S rRNA, tRNAs and other small nuclear and
cytosolic RNAs.

RNA polymerase subunits

Each RNA polymerase has 12 or more different subunits. The largest two subunits
are similar to each other and to the β’ and β subunits of E. coli. RNA polymerase.
Other subunits in each enzyme have homology to the α subunit of the E. coli enzyme.
Five additional subunits are common to all three polymerases, and others are
polymerase specific.

Eukaryotic RNA polymerase activities

Like prokaryotic RNA polymerases, the eukaryotic enzymes do not require a
primer and synthesize RNA in a 5’ to 3’ direction. Unlike bacterial polymerases,
they require accessory factors for DNA binding.

The CTD of RNA Pol II

The largest subunit of RNA polymerase II has a seven amino acid repeat at the C
terminus called the carboxyl-terminal domain (CTD). This sequence,
Tyr-Ser-Pro-Thr-Ser-Pro-Ser, is repeated 52 times in the mouse RNA polymerase II
and is subject to phosphorylation.
rRNA PROCESSING AND RIBOSOMES
Types of RNA processing
In both prokaryotes and eukaryotes, primary RNA transcripts undergo various
alterations of processing events to become mature RNAs. The three commonest
types are: (i) nucleotide removal by nucleases, (ii) nucleotide addition to the 5’- or 3’
-end (iii) nucleotide modification on the base or the sugar.

rRNA processing in prokaryotes

An initial 30S transcript is made in E. coli by RNA polymerase transcribing one of
the seven rRNA operons. Each contains one copy of the 5S, 16S and 23S rRNA
coding regions, together with some tRNA sequences. This 6000 nt transcript folds
and complexes with proteins, becomes methylated and is then cleaved by specific
nucleases (RNase III, M5, M16 and M23) to release the mature rRNAs.

rRNA processing in eukaryotes

In the nucleolus of eukaryotes, RNA polymerase I (RNA Pol I) transcribes the
rRNA genes, which usually exist in tandem repeats, to yield a long, single pre-rRNA
which contains one copy each of the 18S,5.8S and 28S sequences. Various spacer
sequences are removed from the long pre-rRNA molecule by a series of specific
cleavages. Many specific ribose methylations take place directed by small
ribonucleoprotein particles (snRNPs), and the maturing rRNA molecules fold and
complex with ribosomal proteins. RNA Pol III synthesizes the 5S rRNA from
unlinked genes. It undergoes little processing

RNPs and their study

Cells contain a variety of RNA-protein complexes (RNPs). These can be studied
using techniques that help to clarify their structure and function. These include
dissociation, re-assembly, electron microscopy, use of antibodies, RNase protection,
RNA binding, cross-linking and neutron and X-ray diffraction. The structure and
function of some RNPs are quite well characterized.

Prokaryotic ribosomes
Ribosomes are complexes of rRNA molecules and specific ribosomal proteins, and
these large RNPs are the machines the cell uses to carry out translation.The E. coli
70S ribosome is formed from a large 50S and a small 30S subunit. The large subunit
contains 3l different proteins and one each of the 23S and 5S rRNAs. The small
subunit contains a 16S rRNA molecule and 21 different proteins.
Eukaryotic ribosomes
Eukaryotic ribosomes are larger and more complex than their prokaryotic
counterparts, but carry out the same role. The complete mammalian 80S ribosome is
composed of one large 60S subunit and one small 40S subunit. The 40S subunit
contains an 18S rRNA molecule and about 30 distinct proteins. The 60S subunit
contains one 5S rRNA, one 5.8S rRNA, one 28S NA and about 45 proteins
Protein Synthesis, and Degradation
z Protein biosynthesis is achieved by the process of translation.
z During translation, proteins are synthesized on ribosomes by linking amino acids
together in the specific linear order stipulated by the sequence of codons in an
mRNA.
z Ribosomes are the agents of protein synthesis.

z Ribosome Structure and Assembly

¾Ribosomes -- ribonucleoprotein particles found in the cytosol of all cells,
-- as well as in the matrix of mitochondria, and the stroma of
chloroplasts.

z The Composition of Prokaryotic Ribosomes

¾ Escherichia coli ribosomes are representative of the structural organization
of the prokaryotic versions of these supramolecular protein-synthesizing
machines.
¾ E. coli ribosome is a roughly globular particle -- diameter of 25 nm, a
sedimentation coefficient of 70S, and a mass of about 2520 kD.
„ The smaller, or 30S, subunit has a mass of 930 kD and is composed of
--21 different proteins
--16S ribosomal RNA (rRNA) molecule 1542 nucleotides long.

„ The larger 50S subunit has a mass of 1590 kD and consists of

--31 different polypeptides (L1 to L34, see later)
-- two rRNAs, a 2904-nucleotide 23S rRNA and a 120 nucleotide 5S rRNA.
¾ Ribosomes are roughly two-thirds RNA and one-third protein by mass.
¾ An E. coli cell contains around 20,000 ribosomes, constituting about 20% of
the dry cell mass.

z Ribosomal Proteins
-- typically rich in the cationic amino acids Lys and Arg and have few
aromatic amino acid residues, properties appropriate to proteins intended to
interact strongly with polyanionic RNAs.
--Crystal structures obtained thus far for ribosomal proteins reveal a variety of
structures. Because RNA adopts a greater range of structures than
DNA, RNA protein recognition motifs are expected to be diverse.

z rRNAs
--The three E. coli rRNA molecules-23S, 16S, and 5S
--Almost half the bases in 16S rRNA are base paired.Æ generating hairpin
configurations that dominate the molecule; four distinct domains (I through
IV) can be discerned in the secondary structure.

¾Self-Assembly of Ribosomes
--Ribosomal subunit self-assembly is one of the paradigms for the
spontaneous formation of supramolecular complexes from their
macromolecular components.
--the rRNA acts as a scaffold upon which the various ribosomal proteins
convene. Ribosomal proteins bind in a specified order.
¾ Ribosomal Architecture
 --Ribosomal subunits have a characteristic three-dimensional architecture
that has been revealed by image reconstructions from cryoelectron
microscopy and X-ray and neutron solution scattering.
--The small subunit has a channel leading into the cleft, through which
the mRNA passes.
-- in the large subunit with a branched tunnel, supposed the growing
peptidyl chainÆ this tunnel as protein synthesis proceeds.

z Eukaryotic Ribosomes
¾ Eukaryotic cells have ribosomes in their mitochondria (and chloroplasts) as
well as in the cytosol. Æ mitochondrial and chloroplastic ribosomes
resemble prokaryotic ribosomes in size, overall organization, structure, and
function, a fact reflecting the prokaryotic origins of these organelles.
¾ eukaryotic cytosolic ribosomes retain many of the structural and functional
properties of their prokaryotic counterparts, they are larger and considerably
more complex.
¾ higher eukaryotes have more complex ribosomes than lower eukaryotes.
¾ lists the properties of cytosolic ribosomes in a representative mammal, the rat.
--Small (40S) subunits have 33 different proteins and large (60S) subunits
have 49.
--Large subunits have three characteristic rRNAs: 28S, 5.8S, and 5S.

**The sequence of the 5.8S rRNA shows homology to the 5'-end of

prokaryotic 23S rRNA, suggesting it may be an evolutionary derivative of
it.

z The Mechanics of Protein Synthesis

¾ protein biosynthesis in all cells is characterized by three distinct phases:

initiation, elongation, and termination
¾ At each stage, the energy driving the assembly process is provided by
GTP hydrolysis,
¾ specific soluble protein factors participate in the events.
¾ Initiation--involves binding of mRNA by the small ribosomal subunit,
Æ followed by association of a particular initiator
aminoacyl-tRNA (**This codon often lies within the first 30
nucleotides of mRNA spanned by the small subunit). Æ the
large ribosomal subunit then joins the initiation complex,
preparing it for the elongation stage.
¾ Elongation--includes the synthesis of all peptide bonds from the first to the
last.
„ The ribosome remains associated with the mRNA throughout
elongation moving along it and translating its message into an
amino acid sequence.
„ This is a repetitive cycle of events in which successive
aminoacyl-tRNAs add to the ribosome:mRNA complex as
directed by codon binding, and the polypeptide chain grows
by one amino acid at a time.
 „ Only two tRNA molecules are part of the ribosome:mRNA
complex at any moment. Each lies in a distinct site.
‹ A, or acceptor site is the attachment site for an incoming
aminoacyl-tRNA.
‹ P, or peptidyl, site is occupied by peptidyl-tRNA, the tRNA
carrying the growing polypeptide chain.
‹ The elongation reaction transfers the peptide chain from
the peptidyl-tRNA in the P site to the aminoacyl-tRNA
in the A site.
‹ A third tRNA-binding site on the ribosome, the E, or exit,
site, is transiently occupied by deacylated tRNAs as they
exit the P site, having lost their peptidyl chains. Figure
33.6
‹ Elongation is the most rapid phase of protein synthesis.

¾Termination is triggered when the ribosome reaches a "stop" codon (UGA,

UAA, UAG) on the mRNA. At this point, the polypeptide chain is released,
and the ribosomal subunits dissociate from the mRNA.
„ Protein synthesis proceeds rapidly.Æ about 20 a.a. residues / second.
„ So an average protein molecule of about 300 amino acid residues is
synthesized in only 15 seconds. Eukaryotic protein synthesis is
only about 10% as fast.

z Peptide Chain Initiation in Prokaryotes

Æ The components required for peptide chain initiation include (a) mRNA, (b)
30S and 50S ribosomal subunits, (c) a set of proteins known as initiation
factors, (d) GTP, and (e) a specific charged tRNA, f-Met-tRNAfMet.

Initiator tRNA
„ tRNAfMet is a particular tRNA for reading an AUG (or GUG, or even UUG)
codon that signals the start site, it does not participate in chain
elongation,
„ This reaction is catalyzed by a specific enzyme, methionyl-tRNAfMet
formyl transferase. Note that the addition of the formyl group to the
Met-NH2 creates an N-terminal block resembling a peptidyl grouping.
That is, the initiating Met is transformed into a minimal analog of a
peptidyl chain.

mRNA Recognition and Alignment

„ In order for the mRNA to be translated accurately, Æ Recognition of
translation initiation sequences on mRNAs involves the 16S rRNA,
„ the ribosome-binding site, is often called the Shine-Dalgarno
sequence
„ various Shine-Dalgarno sequences found in prokaryotic mRNAs,
along with the complementary 3’-tract on E. coli 16S rRNA.
„ The 3'-end of 16S rRNA resides in the "head' region of the 30S
small subunit.
„ These recognition events are verified by studies of prokaryotic
ribosomes treated with the bacteriocidal protein colicin E3 (a
phosphodiesterase) Æ specifically cleaves the bond after position
 1493, removing the 3'-terminal 49 nucleotides in 16S rRNA that
include the pyrimidine-rich Shine-Dalgarno-binding sequence.

Initiation Factors
„ Initiation involves interaction of the initiation factors (IFs) with GTP,
N-formylMet-tRNAfMet, mRNA, and the 30S subunit to give a 30S
initiation complex
„ the 50S subunit adds to form a 70S initiation complex
„ initiation factorsÆ soluble proteins required for assembly of proper
initiation complexes.

Events in Initiation
„ Initiation begins when a 30S subunit: (IF-3:IF-1) complex binds mRNA
and a complex of IF-2, GTP, and f-Met-tRNAfMet.
„ IF-3 is absolutely essential for mRNA binding Æ not involved in
locating the proper translation initiation site on the message
„ IF-3 also prevets them from reassociating with 50S subunits. IF-3 must
dissociate before the 50S subunit will associate with the mRNA:30S
subunit complex.
„ IF-2 delivers the initiator f-Met-tRNAfMet in a GTP-dependent process
„ IF-2-mediated binding of the initiator tRNA to mRNA and the 30S
subunit.
„ GTP hydrolysis is necessary to form an active 70S ribosome, GTP
hydrolysis is believed to drive a conformational alteration that renders
the 70S ribosome competent in chain elongation.The A site of the 70S
initiation complex is poised to accept an incoming aminoacyl-tRNA.

z Peptide Chain Elongation

The requirements for peptide chain elongation --
(1). an mRNA:70S ribosome: peptidyl-tRNA complex (peptidyl-tRNA in
the P site),
(2). aminoacyl-tRNAs,
(3). a set of proteins known as elongation factors
(4). GTP.

¾ Chain elongation can be divided into three principal steps:

„ Codon-directed binding at the A site (the incoming
aminoacyl-tRNA.)
„ Peptide bond formation: transfer peptidyl chain from the tRNA Æ NH2
group of the new amino acid.
„ Translocation of the "one-residue-longer" These shifts are coupled
with movement of the ribosome one codon further along the mRNA.

Aminoacyl-tRNA Binding
„ EF-Tu binds aminoacyl-tRNA and GTP
„ only one EF-Tu species serving all the different aminoacyl-tRNAs,
„ only in the form of aminoacyl-tRNA:EF-Tu:GTP complexe s,
aminoacyl-tRNAs will bind to the A site of active 70S ribosomes.
 „ Once the aminoacyl-tRNA is situated in the A site, the GTP is hydrolyzed
to GDP and Pi, and the EF-Tu molecules are released as EF-Tu:GDP
complexes.
** GMPPC (nonhydrolyzable GTP analog) permits aminoacyl-tRNA:EF-Tu
binding,Æ EF-Tu does not interact with f-Met-tRNAfMetÆ elongation is
arrested
„ Elongation factor Ts (EF-Ts) promotes Æ recycling of EF-Tu by
mediating the displacement of GDP from EF-Tu and its replacement by
GTP.

Peptidyl Transfer
„ Peptidyl transfer-- transpeptidation, is the central reaction of protein
synthesis, the actual peptide bond-forming step.
„ no energy input is needed;
„ Peptidyl transferase, the activity catalyzing peptide bond formation, is
associated with the 50S ribosomal subunit.

z 23S rRNA Is the Peptidyl Transferase Enzyme

¾E. coli 50S ribosomal subunits from all ribosomal proteins removed Æ retain
significant peptidyl transferase activity.
¾These experiments, carried out by Harry Noller and his colleagues, Æ the
peptidyltransferase enzyme is the 23S rRNA.
¾Nucleotide sequences in this region of 23S rRNA are among the most highly
conserved in all biology.

Translocation
¾ Three things remain to be accomplished in order to return the active 70S
ribosome:mRNA complex to the starting point in the elongation cycle:
„ The deacylated tRNA must be removed from the P site.
„ The peptidyl-tRNA must be moved (translocated) from the A site to the P
site.
„ The ribosome must move one codon down the mRNA so that the next
codon is positioned in the A site.

z GTP Hydrolysis Fuels the Conformational Changes -That Drive Ribosomal

Functions

„ Note that two GTPs are hydrolyzed for each amino acid residue
incorporated into peptide during chain elongation,
--one upon EF-Tu-mediated binding of aa-tRNA
-- one more in translocation.
„ GTP binding induces conformational changes in ribosomal components that
actively engage Æ protein synthesis;
„ GTP hydrolysis and GDP and Pi release relax the system back to the initial
conformational state Æ another turn in the cycle can take place.
„ The energy expenditure for protein synthesis is at least four high-energy
phosphoric anhydride bonds per amino acid. In addition to the two
provided by GTP, two are expended in amino acid activation via
aminoacyl-tRNA synthesis.
z Peptide Chain Termination
„ The elongation cycle of polypeptide synthesis continues until the 70S ribosome
encounters a "stop" codon. At this point, polypeptidyl-tRNA occupies the
P site and the arrival of a "stop" or nonsense codon in the A site signals that
the end of the polypeptide chain has been reached.
„ nonsense codons are not "read" by any "terminator tRNAs" Æ instead
recognized by specific proteins -- release factors, Æ promote polypeptide
release from the ribosome.
„ release factors bind at the A site.
¾RF-1 (36 kD) recognizes UAA and UAG,
¾ RF-2 (41 kD) recognizes UAA and UGA.
**There is about one molecule each of RF-1 and RF-2 per 50 ribosomes.
¾RF-3 (46 kD) function requires GTP Æ binding of RF-1 or RF-2 is
promoted by RF-3
**Ribosomal binding of RF-1 or RF-2 is competitive with EF-G
„ The presence of release factors with a nonsense codon in the A site creates a
70S ribosome : RF-1 (or RF-2) : RF-3-GTP : terrnination signal
complex that transforms the ribosomal peptidyl transferase into a hydrolase
Æ hydrolyzes the ester bond linking the polypeptidyl chain to its tRNA
carrie r(Figure 33.17)

z Polyribosomes Are the Active Structures of Protein Synthesis

„ Active protein-synthesizing units consist of an mRNA with several
ribosomes attached to it Æ such structures are polyribosomes, or, simply,
polysomes.
„ All protein synthesis occurs on polysomes. In the polysome, each ribosome
is traversing the mRNA and independently translating it into polypeptide.
„ In prokaryotes, as many as 10 ribosomes may be found in a polysome;
Eukaryotic polysomes typically contain fewer than 10 ribosomes.

z The Relationship Between Transcription and Translation in Prokaryotes

„ In prokaryotes, ribosomes attach to mRNA even before transcription of the
mRNA is completed, and as a consequence, polysomes can be found in asso-
ciation with DNA.

z Protein Synthesis in Eukaryotic Cells

Eukaryotic mRNAs are characterized by two post-transcriptional modifications:

--the 5' –7methyl-GTP cap
--poly(A) tail.

¾ The 7methyl-GTP cap is essential for ribosomal binding of mRNAs in

eukaryotes and also enhances the stability of these mRNAs by
preventing their degradation by 5'-exonucleases.
¾ The poly(A) tail enhances both the stability and translational
efficiency of eukaryotic mRNAs.
¾ The Shine-Dalgarno sequences found at the 5'-end of prokaryotic
mRNAs are absent in eukaryotic mRNAs.
 z Peptide Chain Initiation in Eukaryotes
¾eukaryotic initiation factors, symbolized as e1Fs.
¾The eukaryotic initiator tRNA is a unique tRNA functioning only in
initiation. Like the prokaryotic initiator tRNA, the eukaryotic version
carries only Met. However, unlike prokaryotic f-Met-tRNAfMet, the Met
on this tRNA is not formylated.Æ the eukaryotic initiator tRNA Æ
designated tRNAiMet, with the "i" indicating "initiation."
„ Eukaryotic initiation can be divided into three fundamental steps.
ÆStep 1: Association of Met-tRNAiMet and initiation factors eIF2,
eIFlA, and eIF3 with the 40S ribosomal subunit to form the
43S preinitiation complex.
**Unlike in prokaryotes, binding of Met-tRNAiMet by
eukaryotic ribosomes is not codon-directed.
ÎStep 2: Binding of the 43S preinitiation complex to mRNA and
migration of the 40S ribosomal subunit to the correct AUG
initiation codon.
--The 43S preinitiation complex binds mRNA at its
5'-terminal 7methyl-GTP cap.
--eIF4E, the mRNA cap-binding protein, represents a key
regulatory element in eukaryotic translation.
--the eIF4E must associate with eIF4G to form a complex
designated e1F4F, eIF4F also contains eIF4A;
--eIF4F binding to the cap structure is a prerequisite for
association of eIF4B and formation of the 48S preinitiation
complex.
--Translation is inhibited when eIF4E binds with 4E-BP
(eIF4E binding protein).Æ Growth factors stimulate
protein synthesis by causing the phosphorylation of 4E-BP,
which prevents its binding to elF4E.
--The 5'-terminal 7methyl GTP cap and the 3'-poly(A) tail act
synergistically to increase translational efficiency.
--Pab1p, the poly(A)-binding protein, has two binding sites,
one for binding to the poly(A) tract on mRNAs and a second
for interaction with eIF4G .
ÆeIF4G serves as a bridge between the cap-binding eIF4E,
the poly(A) tail, and the 40S subunit via elF3. These
interactions initiate scanning of the 40S subunit in search of
an AUG codon.
ÎStep 3: Addition of the 60S ribosomal subunit to the 48S
preinitiation complex, forming the 80S initiation complex,
-- whereupon translation commences.
ÆWhen the 43S preinitiation complex stops at an AUG codon,
GTP hydrolysis in the elF2: Met-tRNAiMet ternary
complex Æ ejection of the initiation factors bound to the
40S ribosomal subunit. Æ release of these factors allows
60S subunit association. eIF2:GDP is recycled to elF2:GTP
by eIF2B; elF2B is a guanine nucleotide exchange factor.

z Regulation of Eukaryotic Peptide Chain Initiation

 ¾Regulation of gene expression can be exerted post-transcriptionally throught
control of mRNA translation. Phosphorylation/dephosphorylation of
translational components is a dominant mechanism for control of protein
synthesis. Peptide chain initiation, the initial phase of the synthetic process,
is the optimal place for such control.

z Peptide Chain Elongation in Eukaryotes

¾ Eukaryotic peptide elongation occurs in very similar fashion to the
process in prokaryotes. An incoming aminoacyl-tRNA enters the
ribosomal A site while peptidyl-tRNA occupies the P site Æ Peptidyl
transfer then occurs Æ followed by translocation of the ribosome one codon
further along the mRNA.
¾ Two elongation factors, EF1 and EF2, mediate the elongation steps.
¾ EF1 consists of two components:
„ EF1A, a 50-kD protein,Æ EF1A is the eukaryotic counterpart of
EF-Tu serves as the aminoacyl-tRNA binding factor and
requires GTP
„ EF1B, a complex of 31-kD (β) and 50-kD (γ) protein subunits,;.
EF1B is the eukaryotic equivalent of prokaryotic EF-Ts Æ
catalyzes the exchange of boound GDP on EF1:GDP for GTP so
active EF1:GTP can be regenerated.
¾ EF2, a 100 kD polypeptide, is the eukaryotic translocation factor. Like its
prokaryotic kin EF-G, EF2 binds GTP, and GTP hydrolysis accompanies
translocation.

z Eukaryotic Peptide Chain Termination

¾ prokaryotic termination involves three different release factors (RFs), just
one RF is sufficient for eukaryotic termination.
¾ Eukaryotic RF (110 kD) is an α2 dimer of 55-kD subunits. Eukaryotic RF
binding to the ribosomal A site is GTP-dependent, and RF:GTP binds at
this site when it is occupied by a termination codon. Then, hydrolysis of the
peptidyl-tRNA ester bond, hydrolysis of GTP, release of nascent
polypeptide and deacylated tRNA, and ribosome dissociation from mRNA
ensue.

z Inhibitors of Protein Synthesis

¾ some of these inhibitors affect prokaryotic but not eukaryotic protein synthesis
and thus are medically important antibiotics.

Streptomycin
„ an aminoglycoside antibiotic Æ affects the function of the prokaryotic 30S
subunit.
„ Low concentrations of streptomycin induce mRNA misreading, so that
improper amino acids are incorporated into the polypeptide. These reading
errors are not frameshift mistakes, Æ susceptible cells are not killed, but
their growth rate is severely depressed.
„ high concentrations, nonproductive 70S ribosome:mRNA complexes
accumulate, preventing the formation of active initiation complexes with
new mRNA.
 Puromycin
„ a structural analog of the aminoacyl-adenylyl grouping characteristic of the
3'-end of aminoacyl-tRNAs.
„ Puromycin binds at the A site of both prokaryotic and eukaryotic ribosomes.
„ Puromycin binding is not dependent on EF-Tu (or EFl).
„ Puromycin serves as an acceptor of the peptidyl chain from peptidyl-tRNA in
the P site, Puromycin aborts protein synthesis through premature termination,
leading to the release of nonfunctional, truncated polypeptides.

Diphtheria Toxin
„ Diphtheria toxin is a phage-encoded enzyme secreted by Corynebacterium
diphtheriae bacteria carrying bacteriophage corynephage β.
„ diphtheria toxin is an NAD+-dependent ADP-ribosylase.
„ One target of diphtheria toxin is the eukaryotic translocation factor, EF2.
„ This protein has a modified His residue known as diphthamide. Diphthamide
is generated post-translationally on EF2; its biological function is
unknown.
„ EF-G of prokaryotes lacks this unusual modification and is not susceptible to
diphtheria toxin.)

Ricin
„ Ricin is an extremely toxic glycoprotein produced by the plant Ricinus communis
(castor bean)
„ The protein is a disulfide-linked, αβ heterodimer;
ÆA subunit (32 kD) is an enzyme and serves as the toxic subunit;
ÆB subunit (33 kD) is a lectin. (Lectins form a class of proteins that bind to
specific carbohydrate moieties commonly displayed by glycoproteins and
glycolipids on cell surfaces.)
„ Endocytosis of bound ricin followed by disulfide reduction releases the A chain,
Æ access to the cytosol and inactivates eukaryotic large ribosomal subunits.
**A single molecule of ricin A chain in the cytosol can inactivate 50,000
ribosomes and kill a eukaryotic cell.
„ Ricin A chain specifically attacks a single, highly conserved adenosine (an A at
position 4256) in the eukaryotic 28S rRNA, Æ inactivate a 60S large
subunit.
„ The adenine in this highly conserved region of the 28S rRNA sequence is
believed to be crucial to functions of the 60S subunit that involve EFl and EF2.

z Protein Folding
¾ Proteins begin to fold even as they are being synthesized on ribosomes.
¾ proteins may be assisted in folding by a family of helper proteins known as
molecular chaperones. Chaperones also serve to shepherd proteins to their
ultimate cellular destinations.
„ The principal chaperones are the Hsp70 and Hsp60 classes of proteins.
„ Hsp70-assisted folding, proteins of the Hsp70 class bind to nascent polypep-
tide chains while they are still on ribosomes. Hsp70 (known as DnaK in E.
coli) recognizes exposed, extended regions of polypeptides that are rich in
hydrophobic residues.
„ A limited number of proteins requires the Hsp60 class of chaperones, which
are known as chaperonins, for the completion of folding
 „ The principal chaperonin in E. coli is the GroES-GroEL complex.
ÆGroEL is made of two stacked 7-membered rings of 60-kD subunits that
form a cylindrical α14 oligomer 15 nm high and 14 nm wide. GroEL has
a 5 nm central cavity that is the site of ATP-dependent protein folding.
ÆGroES consists of a single 7-membered ring of 10-kD subunits that sits
like a dome on GroEL.
„ The eukaryotic analog of GroEL, TRiC, is an 8 or 9-membered double-ring
structure of 55-kD subunits. TRiC lacks a GroES counterpart. Æ the
protein folding is facilitated in an ATP-dependent fashion.

z Post-Translational Processing of Proteins

Î Many proteins must undergo covalent alterations before they become
functional.
Î A survey of some of the more prominent chemical groups conjugated to
proteins, such as carbohydrates and phosphates

Proteolytic Cleavage of the Polypeptide Chain

ÎProteolytic cleavage, as the most prevalent form of protein post-translational
modification, merits special attention.
ÎWhy join a number of amino acids in sequence and then eliminate some of them?
¾ First, diversity can be introduced Met initiating all polypeptide chains,
introduces diversity at N-termini.
¾ Second, proteolysis serves as an activation mechanism The N-terminal
pro-sequence on such proteins may act as an intramolecular chaperone to
ensure correct folding of the active site.
¾ Third, proteolysis is involved in the targeting of proteins to their proper des-
tinations in the cell, a process known as protein translocation.

Protein Translocation
¾Proteins targeted Æ in membranous organelles or Æ export from the cell
Îsynthesized in precursor form carrying an N-terminal stretch of amino acid
residues, or leader peptide, that serves as a signal sequence.
¾signal sequences Æ for sorting and dispatching proteins to their proper
compartments.
Î the information specifying the correct cellular localization of a protein is
found within its structural gene. Once the protein is routed to its destination,
the signal sequence is proteolytically clipped from the protein.
¾a number of eukaryotic membranes are competent in protein translocation,
including the membranes of the endoplasmic reticulum (ER), nucleus,
mitochondria, chloroplasts, and peroxisomes.
¾Several common features characterize protein translocation systems:
„ Proteins to be translocated are made as preproteins containing sorting
signals.
„ Membranes involved in translocation have specific protein receptors
exposed on their cytosolic faces.
„ Translocons, complex structures consisting of several proteins with
different functions, catalyze movement of the proteins across the membrane,
and metabolic energy in the form of ATP, GTP, or a membrane potential is
essential.
 „ Preproteins are maintained in a loosely folded, translocation-competent
conformation through interaction with molecular chaperones.

Prokaryotic Protein Translocation

¾ Gram-negative bacteria typically have four compartments: cytoplasm, plasma
(or inner) membrane, periplasmic space (or periplasm), and outer
membrane.
„ Most proteins destined for any location other than the cytoplasm are
synthesized with amino-terminal leader sequences 16 to 26 amino acid
residues long.
¾ leader sequences, or signal sequences, consist of a basic N-terminal region, a
central domain of 7 to 13 hydrophobic residues, and a nonhelical C-terminal
region.
„ the C-terminal region, include a helix-breaking Gly or Pro residue and
the C-terminal features are not essential for translocation but instead serve
as recognition signals for the leader peptidase, which removes the leader
sequence
„ The exact amino acid sequence of the leader peptide is unimportant.
Nonpolar residues in the center and a few Lys residues at the amino
terminus are sufficient for successful translocation.

Eukaryotic Protein Sorting and Translocation

¾ Eukaryotic cells are characterized by many membrane-bounded compartments.
¾ In general, signal sequences targeting proteins to their appropriate compart-
ments are located at the N-terminus as cleavable presequences, although many
proteins have internal, noncleaved targeting sequences.
¾ Proteolytic removal of the leader sequences is also catalyzed by specialized
proteases, but removal is not essential to translocation.

The Synthesis of Secretory Proteins and Many Membrane Proteins

Is Coupled to Translocation Across the ER Membrane
„ In higher eukaryotes, translation and translocation of many proteins destined for
processing via the ER are tightly coupled. Î translocation across the ER
occurs as the protein is being translated on the ribosome.
„ the N-terminal signal sequence of a preprotein undergoing synthesis emerges
from the ribosome, it is detected by a so-called signal recognition particle
(SRP) .
„ SRP is a 325-kD nucleoprotein assembly that contains six polypeptides and a
300-nucleotide 7S RNA.
„ SRP-ribosome complex then diffuses to the cytosolic face of the ER,Æ binds to
the docking protein (also known as the SRP receptor), a heterodimeric
proteinÆ α-subunit -- anchored to the membrane by the transmembrane
β-subunit; Î both subunits have GTPase activity. Docking is followed by
dissociation of SRP in a GTP-dependent process.
„ After targeting to the membrane Æ delivering its growing polypeptide to the
translocon.
ÎThe translocon is a complex, multifunctional entity that includes, among
other proteins, the SRP receptor and Sec61p, a heterotrimeric complex of
membrane proteins (Sec61p is known as SecYEGp in prokaryotes).
 „ Sec61p has 10 membrane-spanning segments Æ Sec61p serves as the
transmembrane channel Æ the nascent polypeptide is transported into the ER
lumen. The pore size of Sec61p is about 2 nm.
„ Soon after it enters the ER lumen, the signal peptide is clipped off by
membrane-bound signal peptidase.
„ Other modifying enzymes within the lumen introduce additional
post-translational alterations into the polypeptide, such as glycosylation with
specific carbohydrate residues.
„ ER-processed proteins destined for secretion from the cell or inclusion in
vesicles such as lysosomes
„ polypeptides Æ membrane proteins Æ carry 20-residue hydrophobic
stop-transfer sequences within their mature domainsÆ arrest their passage
across the ER membrane.

Mitochondrial Protein Import

„ Most mitochondrial proteins are encoded by the nuclear genome and
synthesized on cytosolic ribosomes.
„ Mitochondria consist of three principal subcompartments: the outer membrane,
the inner membrane, and the matrix.
„ mitochondria proteins must not only find mitochondria, they must gain access to
the proper subcompartment, and once there they must attain a functionally
active conformation.
„ Signal sequences on nuclear-encoded proteins destined for the mitochondria are
N-terminal cleavable presequences 10 to 70 residues long. These
mitochondrial presequences lack contiguous hydrophobic regions. Instead,
they have positively charged and hydroxyl-amino acid residues spread along
their entire length. These sequences form amphiphilic α-helices
„ In general, mitochondrial targeting sequences share no sequence homology.
Once synthesized, mitochondrial preproteins are retained in an unfolded state
with their target sequences exposed, through association with heat shock
proteins of the Hsp70 family. Import involves binding of a preprotein to a
receptor protein on the mitochondrial outer membrane and subsequent uptake
via the mitochondrial protein translocation apparatus

z Protein Degradation

„ Protein degradation poses a real hazard to cellular processes. To control this

hazard, protein degradation is compartmentalized, either in macromolecular
structures known as proteasomes or in degradative organelles such as
lysosomes.
„ The proteasome is a functionally and structurally sophisticated counterpart
to the ribosome. Regulation of protein level via degradation is an essential
cellular mechanism. Regulation by degradation is both rapid and
irreversible.

The Ubiquitin Pathway for Protein Degradation in Eukaryotes

„ Ubiquitination is the most common mechanism to label a protein for. some

degradation in eukaryotes.
 „ Ubiquitin is a highly conserved, 76-residue (8.5 kD) polypeptide
widespread in eukaryotes. Proteins are condemned to degradation through
ligation to ubiquitin.
„ Three proteins in addition to ubiquitin are involved in the ligation
process: El, E2, and E3
„ E1 is the ubiquitin-activating enzyme (105-kD dimer). It becomes
attached via a thioester bond to the C-terminal Gly residue of ubiquitin
through ATP-driven formation of an activated ubiquitin-adenylate
intermediate.
„ Ubiquitin is then transferred from E1 to an SH group on E2 the
ubiquitin-carrier protein.
„ E3 (180 kD), the ubiquitin-protein ligase. E3 plays a central role in
recognizing and selecting proteins for degradation.
„ Other proteins targeted for ubiquitin ligation and proteasome degradation
contain PEST sequences-short, highly conserved sequence elements in
proline (P), glutamate (E), serine (S), and threonine (T) residues.

Proteasomes

„ Proteasomes are large oligomeric structures enclosing a central cavity

where proteolysis takes place.
„ The 20S proteasome barrel is about 15 nm in height and 11 nm in diameter
and contains a three-part central cavity. These rings are believed to unfold
proteins destined for degradation and transport them into the central cavity.
The 8-subunits possess the proteolytic activity. The products of proteasome
degradation are oligopeptides 7 to 9 residues long.
„ Eukaryotic cells contain two forms of proteasomes: the 20S proteasome,
and its larger counterpart, the 26S proteasome. The eukaryotic 26S
proteasome (1700 kD) is a 45-nm-long structure composed of a 20S
proteasome plus 2 additional substructures known as 19S caps or PA700
(for proteasome activator-700 kD).
Regulation of Gene Expression
The cellular concentration of a protein is determined by
a delicate balance of at least seven processes, each
having several potential points of regulation:

1. Synthesis of the primary RNA transcript

(transcription)
2. Posttranscriptional modification of mRNA
3. Messenger RNA degradation
4. Protein synthesis (translation)
5. Posttranslational modification of proteins
6. Protein targeting and transport
7. Protein degradation

¾ housekeeping genes
Genes for products that are required at all times, such as those for the enzymes
of central metabolic pathways, are expressed at a more or less constant
level in virtually every cell of a species or organism.
¾ Unvarying expression of a gene is called constitutive gene expression. For other
gene products, cellular levels rise and fall in response to molecular signals; this is
regulated gene expression.

¾ RNA polymerase binds to DNA at promoters

Promoter region

Transcription initiation is regulated by proteins- that bind to or near promoters

At least three types of proteins regulate transcription initiation by RNA polymerase:
• specificity factors alter the specificity of RNA polymerase for a given promoter or
set of promoters;
• repressors impede access of RNA polymerase to the promoter;
• activators enhance the RNA polymerase–promoter interaction.
Gene expression :
Gene expression 包含下列幾個步驟
•transcription level – gene ÆmRNA
•translational level – mRNA Æ protein
•posttranslational level Æ protein active or inactive
¾基因依其功能可分為 Æ regulator gene； structure gene
¾基因調節原則 ─ 當生物體需要此基因產物時則，基因表現 Æ turn on 反之
則 Æturn off
¾bacterial and phage 基因的 turn on /off 主要是藉由 transcription 來進行
regulation (調節)
¾positive regulator -- active some enzyme Æ induce gene transcription Æ gene
 expression
¾negative regulator -- active some enzyme Æ inhibit gene transcription Æ no
gene expression 基因的調節可分為：正向調節和負向調節 （如下圖所示）

A map of the lactose operon：

Operon models - The original Jacob--Monod model for gene regulation, based upon the
lactose operon system, is shown schematically . A more complete description of the lac
operon is shown in Figure Transcription of the three structural genes is initiated near an
adjacent site, the operator. Transcription yields a single polycistronic messenger RNA
(that is, an RNA containing all three genes). The term cistron is used here to indicate a
region of a genome that encodes one polypeptide chain.

z Several DNA-binding motifs have been described that play prominent roles in the
binding of DNA by regulatory proteins: helix-turn-helix, zinc finger and
homeodomain (a type of DNA-binding domain found in some eukaryotic
proteins)
¾ Helix-Turn-Helix This DNA-binding motif is crucial to the interaction of
many prokaryotic regulatory proteins with DNA, and similar motifs occur in
some eukaryotic regulatory proteins. The helix-turn-helix motif comprises
about 20 amino acids in two short -helical segments, each seven to nine
amino acid residues long, separated by a turn
¾ Zinc Finger In a zinc finger, about 30 amino acid residues form an elongated
loop held together at the base by a single Zn2 ion, which is coordinated to
four of the residues (four Cys, or two Cys and two His).
¾ Homeodomain Another type of DNA-binding domain has been identified in
a number of proteins that function as transcriptional regulators, especially
 during eukaryotic development. This domain of 60 amino acids—called the
homeodomain

z Regulatory Proteins Also Have Protein-Protein Interaction Domains: Two

important examples are the leucine zipper and the basic helix-loop-helix.
¾ Leucine Zipper This motif is an amphipathic helix with a series of
hydrophobic amino acid residues concentrated on one side

¾ Basic Helix-Loop-Helix Another common structural motif occurs

in some eukaryotic regulatory proteins implicated in the control of
gene expression during the development of multicellular organisms.
These proteins share a conserved region of about 50 amino acid
residues important in both DNA binding and protein dimerization.
This region can form two short amphipathic helices linked by a
loop of variable length, the helix-loop-helix (distinct from the
helix-turn-helix motif associated with DNA binding). The
helix-loop-helix motifs of two polypeptides interact to form
dimmers.

Regulation of Gene Expression in Prokaryotes

z Lactose Operon Regulation:
Operon composition/induction - The lactose operon consists of three linked
structural genes that encode enzymes of lactose utilization, plus adjacent regulatory
sites. The three structural genes--z, y, and a--encode β-galactosidase, β-galactoside
permease (a transport protein), and thiogalactoside transacetylase (an enzyme of
still unknown metabolic function), respectively.
¾ In the presence of an inducer, all three enzymes accumulate simultaneously, but to
different levels. Lactose itself leads to induction of the lactose operon (also
called the lac operon), but the true intracellular inducer is allolactose
(Gal (1-->6)Glc), a minor product of β-galactosidase action.
¾ In the laboratory one usually uses a synthetic inducer such as
isopropylthiogalactoside (IPTG), which induces the lactose operon but is not
cleaved by β-galactosidase. Hence, its concentration does not change during an
experiment.

z Lac Repressor
Lac repressor properties - The lac repressor was isolated in 1966 by Walter
Gilbert and Benno Müller-Hill.
Æ lac repressor is a tetramer, formed from four identical subunits, each with 360
amino acids (Mr = 38,350).
Æ The protein binds isopropylthiogalactoside (IPTG, a synthetic inducer) with a
Ka of about 106 M-1, and it binds nonspecifically to duplex DNA with a Ka of
about 3 x 106 M-1. However, its specific binding at the lac operator is much
tighter, with a Ka of 1013 M-1.
 Æ Lac repressor binding of operator - Control by the lac repressor is
exceedingly efficient, particularly in view of the minute amount of repressor
present in an E. coli cell. The i gene is expressed at a very low rate, to give
about 10 molecules of repressor tetramer per cell.

z Many genes for amino acid biosynthetic enzymes are regulated by

transcription attenuation
@ trp operon
function: trp (tryptophan) 合成。
Trp operon 組成： 5 structure genes:
trpE、trpD、trpC、trpB、trpA
control element: promoter – contain
operator
Trp L: L 代表引導序列 (leader) ,
內含 trp a，a 代表 attenuator
trp R regulate trp operon : trp R
aporepressor; when bind to trp active
repressor this complex bind to
operator turn off
** inducible operon – with inducer
turn on the operon (positive control)
lac operon, ara operon
**repressible operon – negative
control trp, his operon

z Induction of the SOS Response Requires Destruction of Repressor Proteins

The SOS response is acvated – replication fork stalls

Æ RecA:DNA complex binds into LexA
Æ LexA conformational change
Æ LexA cleave itself
Æ mutasome form, (an error-prone replication
apparatus )
Æ DNA polymerase to replicate past the lesion.

** LexA protein blocks expression of many genes

encoding a set of proteins mediating error-prone
replication.

z Eukaryotes have at least three main mechanisms of translational regulation.

„ Initiation factors are subject to phosphorylation by a number of protein
kinases. The phosphorylated forms are often less active and cause a general
depression of translation in the cell.

„ 2. Some proteins bind directly to mRNA and act as translational repressors,

many of them binding at specific sites in the 3 untranslated region (3UTR).
So positioned, these proteins interact with other translation initiation factors
bound to the mRNA or with the 40S ribosomal subunit to prevent
translation initiation.
„ 3. Binding proteins, present in eukaryotes from yeast to mammals, disrupt
the interaction between eIF4E and eIF4G. The mammalian versions are
known as 4E-BPs (eIF4E binding proteins). When cell growth is slow, these
proteins limit translation by binding to the site on eIF4E that normally
interacts with eIF4G. When cell growth resumes or increases in response to
growth factors or other stimuli, the binding proteins are inactivated by
protein kinase-dependent phosphorylation.