CHEM 3320H - Metabolism

See the posted syllabus on Blackboard

Come to your lab this week

Lecture 1: Metabolism overview and recurring chemistry in metabolism

• Protein structure
• Enzyme kinetics
• Enzyme mechanisms
• The roles of cofactors
• Cooperativity and enzyme regulation
• Free energy changes (ΔG) of reactions

What you have been introduced to in CHEM 3310H will be applied and expanded on in
CHEM 3320H, and repeatedly so.
Image source: Futurama Season 3 Episode 17, A Pharoah to Remember.
 Plan for Success

• Attend the lectures: not everything that is relevant lends

itself to Powerpoint.
• Write the structures and write the paths
• Key points may not be obvious from Powerpoint alone.
• Do the posted work sets in class when I am there to help.
Timely help may not be available otherwise.
• Certain topics rely on literature review papers, which you
may not fully understand without engaging in questions
and answers during class
The panel above is a wall poster of metabolism. There is a copy down the E corridor of the
Chemical Sciences Building. You can download a high-resolution version from the
course Blackboard site.
In CHEM 2300H much of the focus was on the biomolecules that life is composed of. In
CHEM 3310H our focus turned to how some of these biomolecules, namely enzymes,
could enhance the reaction rates of processes of biochemical importance. This focus,
however, was very narrow; we we interested in explaining how a particular enzyme (such
as chymotrypsin) worked to a catalyze specific reaction (hydrolysis of a protein to
peptides). But within a cell, thousands of such reactions will be occurring, and they will
be doing so with a purpose: the continued existence of the cell or organism. These
reactions - their reactants, products and intermediates; their enzymes; their regulators;
their thermodynamics – are what constitute metabolism. This is the focus of CHEM
3320H.
 Catabolism and anabolism: the two arms of metabolism

1. CATABOLISM 2. ANABOLISM
Complex molecules ADP
Complex molecules
(eg glucose) + Pi (eg amino acids)

Simple molecules
ATP Simple molecules
(eg CO2, H2O)
(eg CO2, H2O, NH3)

Catabolism refers to those processes that provide energy for ATP formation, typically
from the breakdown of complex biomolecules to simpler ones, while anabolism refers to
the processes that use the energy of ATP to synthesize complex biomolecules from
simpler precursors. As shown below in the time-lapse picture of bacterial growth, both
arms of metabolism occur simultaneously: the bacteria take in nutrients from their
surroundings, use these to extract energy to form ATP, some of which is used to build up
their own biomolecules required for cell growth and division.
In addition, we will see that there are pathways that can be used for both anabolism and
catabolism. These are called amphibolic pathways.
 How is metabolism controlled?

• Control of enzyme levels

• Control of enzyme activity
• Compartmentalization of pathways in
different organelles
• Metabolic channeling

Control of enzyme levels is a matter of molecular biology, as this is an example of control

by gene expression, which is beyond the scope of this course
Control of enzyme activity is a topic that has been introduced in previous biochemistry
courses, and we will expand on it in CHEM 3320H.
Compartmentalization touches on cell biology, but it has an important biochemical
component. Thousands of reactions occur within the cell, and we will see that certain
reactions are often associated with specific locations within the cell. Metabolic control
can be exerted in part by controlling the export of a metabolite from a location where it is
made to a location where it is further processed. This typically involves considerations of
transport across membranes.
More recently, metabolic channeling is now being recognized as another way for
controlling metabolism. This is the physical association of enzymes that constitute a
metabolic pathway. The pyruvate dehydrogenase complex ia one such example that we
will deal with, and there is evidence that other pathways have such channeling too,
which we will likely deal with towards the end of the course.
Image source: https://www.deviantart.com/monstara/art/Animal-cell-49149164
 Stages of metabolism
Catabolic
Anabolic
Stage1 Stage 2 Stage 3

Complex biomolecules Monomers Metabolites Inorganic precursors

eg Proteins eg amino acids eg pyruvate eg water
Polysaccharides monosaccharides acetyl CoA carbon dioxide
RNA & DNA nucleotides citric acid cycle intermediates ammonia
Phospholipids fatty acids urea cycle intermediates

Good news! Key metabolic processes are usually similar or identical in many different organisms

As in the previous panel, red arrows indicate catabolic processes (which release energy
in a biologically useful form) while blue arrows denote anabolic ones (which use this
energy to drive their reactions). This panel shows some more detail than the previous
panel, and divides metabolic processes into stages according to the complexity of the
molecules involved.
At the top are the complex biomolecules that make up much of an organism: its
proteins and enzymes of various functions, polysaccharides (starch, glycogen, cellulose,
chitin, and the sugar component of glycoproteins and glycolipids), nucleic acids of its
genetic material and gene expression (DNA, RNA) and the complex lipids that are used to
make cell membranes. Stage 1 of catabolism is the breakdown of these complex
molecules to their component monomers, while Stage 1 of anabolism is the
condensation of these monomers to make the more complex biomolecules. Some of the
Stage 1 anabolic processes you have studied before in other courses, such as molecular
biology (replication, transcription, translation). You also have some experience with
Stage 1 catabolic processes, such as the hydrolysis of proteins by proteases.
In CHEM 3320H our focus will be on Stage 2 catabolic and anabolic reactions, and
Stage 3 catabolic reactions. (We can do no more than this as there are not enough hours
in the class). A major part of what we will be studying are the metabolic intermediates
listed above, also known as metabolites. Like ATP, metabolites are typically not present
at high concentrations within the cell (typically ~ 10 μM to 10 mM, depending on the
molecule), but there is a high flux through them as they are constantly being made and
consumed. Metabolites can be converted to the inorganic molecules shown above,
which releases more energy for ATP formation. Alternatively, they can be used to
assemble other molecules. For example, we will see that pyruvate produced by the
catabolism of sugars can be used to synthesize amino acids such as alanine.
 Central metabolism is our first topic

Citric Acid
Cycle
Gluconeogenesis PDC
Glucose Pyruvate Acetyl CoA CO2
Glycolysis

Polysaccharides reduced oxidized

(glycogen, starch) redox cofactors redox cofactors

ADP + Pi
Oxidative
Phosphorylation
ATP
O2 H2O

Central metabolism is so named because all other branches of metabolism, sooner or

later, converge on it at some point. An outline of central metabolism is shown above.
Red arrows and blue arrows indicate catabolic and anabolic processes, respectively.
Some pathways, like the citric acid cycle, can be used for both anabolism and
catabolism, and that is indicated by its purple colour. Dotted arrows indicate electron
flow as substrates are oxidized and redox-active (NAD+ and FAD) are reduced.
Glycolysis is perhaps the most ancient and universal metabolic pathway. In it, glucose, a
six-carbon sugar, is oxidized to a pair of three-carbon pyruvate molecules, accompanied
by the capture of some energy as ATP. NAD+ is the oxidizing agent, and is reduced to
NADH.
For aerobic organisms such as humans, pyruvate can be further catabolized, releasing
far more energy than can be obtained by glycolysis alone. This involves three processes:
1. Oxidation of pyruvate to acetate (as a thioester to Coenzyme A) by the pyruvate
dehydrogenase complex (PDC) reduces NAD+ to NADH.
2. Oxidation of acetate to CO2 by the citric acid cycle reduces NAD+ and FAD to NADH
and FADH2.
3. Oxidation of NADH and FADH2 by O2 is highly favorable and the free energy of this
process is captured by the respiratory chain and is used to drive phosphorylation of
ADP by Pi. This process is called oxidative phosphorylation. The resulting oxidized
cofactors NAD+ and FAD are then reduced again by further action of glycolysis, the
PDC, and the citric acid cycle.
So much for the overview; we will soon start filling in the molecular details. We will also
examine fatty acid metabolism, which converges with central metabolism at acetyl CoA,
and amino acid metabolism, which involves the citric acid cycle and other processes.
 Gluconeogenesis: Synthesis of glucose from pyruvate

ADP, Pi ATP

Glycolysis
Q1. Is gluconeogenesis simply
Glucose Pyruvate glycolysis in reverse?
Q2. Why not?
Gluconeogenesis

ADP, Pi ATP

We will also consider the synthesis of glucose from pyruvate by the process of
gluconeogenesis. This will also be the point in which we see that metabolic pathways
that run in opposite directions are not simply the identical pathway run backwards. It will
also be the point at which we consider reciprocal regulation of pathways. It would make
no sense of a cell to be running glycolysis and gluconeogenesis at the same time, and
these processes are regulated so that such a futile cycle such as the one shown above
will not occur.
 Metabolic redox reactions rely on NAD+ and NADP+

NAD+ NADP+

Whenever we consider a redox reaction (the transfer of electrons) we will in most cases
be considering reactions that use the nicotinamide cofactors NAD+ and NADP+ as
electron carriers. The above diagram shows how NAD+ is the main carrier of electrons in
catabolism (left half), while NADP+ acts in anabolism (right). In addition, redox balance
can be maintained through the action of transhydrogenase (bottom of figure).
 What’s the chemistry behind these pathways?

Mainly that of simple organic functional groups:

• Reactions of alcohols and amines
• Reactions of aldehydes and ketones
• Reactions of esters and carboxylic acids
• Reactions of amines
• Plus a few more
Recurring chemistry in biochemistry: common electrophiles and their reactions

Carbonyl Compounds

Aldehydes Ketones Esters Thioesters Protonated Imines Phosphate

δ- δ- δ- δ- δ- δ-
O O O O RNH+ O

C C C δ- C δ- C -O P δ-
R δ+ H R δ+ R R δ+ OR R δ+ SR R δ+ R δ+ O-
-O

O O-

Such electrophiles react C C

readily with nucleophiles (Nu:) R R R R
Nu
Nu:

The carbonyl groups of aldehydes, ketones, and esters are polarized, as indicated by the
slight negative charge on the carbonyl oxygen (δ-) and the slight positive charge on the
carbonyl carbon (δ+). Consequently these carbonyl group are electrophiles and they
readily react with nucleophiles (molecules with a lone pair of electrons). Nucleophiles,
which provide a lone pair of electrons for reactions are generically represented by the
term Nu: as shown above. For similar reasons, protonated imines are also electrophiles,
as is phosphate (Pi) and protons
 Common biochemical nucleophiles

Nucleophiles Nu: Are electron pair donors R

N

HO- R-O- R-S- N

alkoxides thiolates H
hydroxide
(eg deprotonated Ser) (eg deprotonated Cys) imidazole
(eg deprotonated His)

R3-C-
resonance-stabilized carbanions
(examples to come)
 Acyl groups and nucleophilic acyl substitutions

As seen in CHEM 3310H with proteases and esterases!

An acyl group is a carbonyl compound that is also bonded to an electronegative atom

such as O, S, or N. Above, the acyl group is circled within the dotted lines.
Acyl groups react readily with nucleophiles in substitution reactions. In fact, you’ve seen
such a reaction before, as the hydrolysis of a peptide bond (an amide) is an example.
 Aldehydes, ketones and nucleophilic addition reactions

Aldehydes and ketones have carbonyl groups too, but these are adjacent to a carbon or a
hydrogen and they do not undergo acyl substitution reactions like those shown on the
previous slide. Their tendency is to undergo addition reactions as shown above. The
reaction flagged with the star is that which generates the ring structures of simple
sugars, and we’ll see other examples later.
 Resonance-stabilized carbanions from carbonyl compounds & their reactions

:Base = B:, any group that can act as a proton acceptor

Certain nucleophiles are common biochemical bases:

Resonance-stabilized enolate anion

Usually it is difficult to remove a proton from a carbon atom, as the C-H bond is strongly
covalent. However, an important exception is the carbon that is next to a carbonyl group
(called the alpha carbon). As the product is resonance-stabilized, it is relatively easy to
remove the proton on an alpha carbon. This step is important in further reactions that
form carbon carbon bonds in anabolic processes. Active-site groups that can act as
bases are side chains of Glu, Asp, and His. Remember also that in catalysis if am active
site-group acts as a proton acceptor (base) in one part of the reaction cycle, it has to act
as a proton donor (acid) in another part of the cycle.
Resonance-stabilized enolates and carbon-carbon bond formation

Resonance-stabilized enolate as a nucleophile

Claisen condensation:
Aldol condensation: Reaction of an enolate
Reaction of an enolate (nucleophile)
(nucleophile) with an ester
with an aldehyde/ketone (electrophile)
(electrophile)

These reactions may be familiar to you from organic chemistry, and they also occur in
anabolic pathways in biochemistry. The resonance-stabilized enolate anion that was
generated by deprotonation of the alpha carbon of a carbonyl compound can act as a
nucleophile and add to an electrophilic carbonyl of an aldehyde/ketone as in an aldol
condensation, or to an ester in the case of a Claisen condensation. We’ll see examples
of both of these during the course.
 :Base = B:
Elimination reactions in metabolism

Dehydration of beta-hydroxy carbonyl compounds Alcohol oxidation by NAD+

Hydride (H-) as a leaving group

Elimination reactions are also common in metabolism. These typically begin with a base
removing an acidic proton.
As on the previous two panels, we see yet again, on the left, a reaction that starts with
the deprotonation of an alpha carbon in a carbonyl compound, generating a resonance-
stabilized enolate. If this is a beta-hydroxy compound, loss of water results in a double
bond between the carbons that are alpha and beta to the carbonyl group. We will see
such a reaction and its reverse in fatty acid metabolism.
On the right, is an example of a reaction that you have seen before involving NAD+ acting
to oxidize an alcohol. Many such reactions in NAD+ -dependent dehydrogenases are
examples of elimination reactions.
 See the posted document
ORGANIC FUNCTIONAL GROUPS AND REACTIONS IN METABOLISM
1. Drawing relevant and correct chemical structures Page 2
Chemical & Biochemical Shorthand
2. Covalent and noncovalent bonds in biochemistry Page 6
The size scale of biochemistry
The bond energy scale of biochemistry
Resonance structures.
Electronegative atoms and polarized covalent bonds
Hydrogen Bonds
3. Common Organic Reaction Mechanisms in Central Metabolism Page 11
Protonation and deprotonation reactions
Hydride transfer
Resonance structures.
Tautomers
The role of carbonyl groups in biochemistry
- Resonance stabilization of carbanions.
- Formation of hemiacetals & hemiketals.
- Reactions of hydroxy carbonyls compounds.
Thiamine pyrophosphate: when you need an electron sink but don't have one

In the folder for this lecture is a document that will help you get up to speed with the
organic chemistry that you’ll need to succeed in this course. It includes some samples
you can work on. Refer to this document frequently as you progress through the course