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OUTLINE Primary Structure

ENZYMES 1 ● Describes not only the number and identity of amino acids in a
- Enzyme structure protein but more importantly, the order or sequence to which
- Terms to remember
- Enzyme Classification and Nomenclature 2 specific amino acids combine.
- Classes of Enzymes
- EC Code and Systematic Name
- Enzyme Kinetics 3
- Activation Energy
- Types of Specificity
- Factors that influence enzymatic reactions
- Substrate concentration 4
- Michaelis Menten Constant (Km)
- Measurement of Enzyme Activity 5 Secondary Structure
- Enzymes as reagents ● Since proteins or polypeptides of enzymes contain an amino group
Creatinine Kinase in one end and a carboxyl group in the other end, one polypeptide
Lactate Dehydrogenase 7
- Sample Electrophoregram 8 can twist and bind with each other by hydrogen bond.
- Correspondence between CPK and LDH isoenzyme patterns ● This twisting actually forms its secondary structure.
Aspartate aminotransferase (AST) ● Enzymes can wrap around to form alpha helix or twist on top of
Alanine aminotransferase (ALT) 9
themselves to form beta sheets (plated sheet).
Alkaline phosphatase (ALP)
Acid phosphatase (ACP) 11 ● If this structure will further fold, it will form the tertiary structure of the
Gamma glutamyl transferase (GGT) enzyme.
Amylase (AMS) 12
LIpase (LPS) 13
Glucose-6-Phosphate Dehydrogenase (G6PD)
Cholinesterase (CHS)
Aldolase (ALS) 14
Ornithine Carbamoyl Transferase (OCT)
Leucine aminopeptidase (LAP)
LEGEND
BLACK TEXT COLORED TEXT
Based from ppt Based from lecture proper
Tertiary Structure
● It is the 3 dimensional shape of the entire polypeptide chain which
Intended Learning Outcomes involves folding.
At the end of the unit, students should be able to:
1. compare and contrast the various clinically significant enzymes
and the methods of used to measure each enzyme
2. interpret laboratory results to check for its reliability and also to
assess diseases associated with abnormal levels of each analyte
3. create an efficient way of managing laboratory works and
demand for shortened turn around time for certain laboratory
tests
4. apply laboratory safety procedures and precautions in the
performance of laboratory measurements both in the clinical Quaternary Structure
laboratory and in the community ● Take note the NOT ALL enzymes have quaternary structure.
● This only exists when a protein or enzyme has more than one
Enzymes polypeptide present.
● Comes from the Greek word “enzymos” (?) meaning leaven or in ● spatial relationship
yeast.
● First discovered by Louis Pasteur, Wilhelm Kuhne, and Eduard
Buchner in the 1800s.
● What are Enzymes?
○ Biologic proteins
■ All enzymes are proteins but not all proteins are enzymes.
○ Catalyze chemical reactions
■ Catalyze: accelerate the rate or speed up chemical Terms to remember
reactions in the body.
Substrate ● Substance acted upon by the enzyme.
■ During the reaction, they do not become chemically
altered nor destroyed nor consumed in the reaction. Active Site ● Water free cavity, where the substrate interacts
○ Important for physiologic functions with particular charged amino acid residues
○ Found in all body tissues ● Found inside the enzyme.
Allosteric Site ● A cavity other than the active site; bind the
Enzyme Structure regulator molecules.
● Globular in nature. ● Found inside the enzyme.
● They have an area in its surface which can only bind a specific
substance which we call the substrate to which the enzyme can How does an enzyme look during interaction?
only accept. ● The reaction of enzyme to substrate may
● The binding leads to changes in both molecules which result in the be described as similar to a lock
formation of the required product and also restoration of enzyme to (enzyme) and key (substrate).
its original state. ● In the enzyme we have 2 sites, the active
● Enzyme will not be destroyed nor changed but will be recovered at site and the allosteric site.
the end of the reaction and is ready to take another substrate ● Here the substrate binds at the active site which starts the reaction.
molecule.
Isoenzymes or ● Although enzymes share similar or the same
Isozymes catalytic function throughout the body, some
 enzymes may exist in different forms within the ● Since we have numerous enzymes, it is necessary to make sure that
same individual. the nomenclature is standardized.
● Enzymes with the same function but exist in ● This standardization of enzyme nomenclature was devised by the
different forms. Enzyme Commission of the International Union of Biochemistry
○ These different forms may be in 1961, and revised in 1972 and 1978.
differentiated from each other based on ● Nomenclature include:
certain physical properties such as ○ class of enzymes,
electrophoretic mobility, solubility, or
○ EC code (Enzyme Classification Code),
resistance to activation.
● Differ in amino acid sequence or in protein ○ systematic name which defines the substrate acted on,
structures. ○ reaction catalyzed and possibly the name of coenzyme
● Although they have different forms, they involved in the reaction, and
actually have the same 3D structure. ○ because systematic names are usually very long, the IUB or
● (same with isoforms) Contributes to the the Enzyme Commission also provided a more usable common
heterogeneity in the properties and functions of
name or recommended name
enzymes.
Isoforms ● Enzymes that have been subjected to Classes of Enzymes
post-transcriptionally modified. ● Arranged in this order and should not be interchanged.
● (same with isozymes) Contributes to the
heterogeneity in the properties and functions of 1 Oxidoreductases → Functions to catalyze oxidation reduction
enzymes. reactions.

Cofactor ● non-CHON (nonprotein) molecules of enzymes 2 Transferases → Catalyze the transfer of one group of a
necessary for enzyme activity compound to another.
3 Hydrolases → Catalyzes hydrolysis of bonds.
Activators: inorganic factors 4 Lyases → Catalyzes the removal of a group without
● Chloride, bromide, magnesium, iron, zinc, hydrolysis and the products contain double
copper, calcium, manganese, cobalt, bonds.
potassium, amd etc.
5 Isomerases → Catalyzes the interconversion of geometric,
Coenzyme: organic cofactors optical, or positional isomers.
● Coenzyme A, folic acid coenzyme, thiamine
pyrophosphate, cobamide coenzyme, 6 Ligases → Catalyze the joining of two substrate
nicotinamide coenzymes, biotin, flavin molecules coupled with breaking of
coenzymes, lipoic acid, coenzyme Q, pyridoxal pyrophosphate bond and adenosine
phosphate, and etc. triphosphate (ATP) or a similar compound.
Prosthetic ● a coenzyme bound tightly to the enzyme
Group EC Code and Systematic Name
● Enzyme Commission Code consists of 4 digits.
Apoenzyme ● The enzyme portion bound to the prosthetic
○ 1st digit: class (1-6)
group
○ 2nd digit: subclass
Holoenzyme ● Prosthetic group (coenzyme) + apoenzyme ○ 3rd digit: sub-subclass
(enzyme) ○ 4th digit: serial number in its sub-subclass
● inactive form of the enzyme ● Example:
Proenzyme or
Zymogen ● Not all the time, enzymes are in their active ○ Transferase
forms. Sometimes we need to make them ■ AST/SGOT 2.6.1.1 L-aspartate:2-oxaloglutarate
active. aminotransferase
○ Most digestive enzymes are inactive. They
are not always active. This will prevent Class: 2. Transferases
digestive enzymes from digesting their Reaction: transfer of amino group
places of synthesis if they are always Systematic Name: L-aspartate:2-oxaloglutarate aminotransferase
active. Hence, they are inactive and only Substrate: L-aspartate
are activated when needed. Coenzyme: ----
Recommended Name: AST
SGOT (older term)

○ Oxidoreductase
■ LD/LDH 1.1.1.27 L-lactate:NAD+ oxidoreductase
Class: 1. Oxidoreductase
Reaction: catalyzing oxidation reduction method (RedOx)
Systematic Name: L-lactate:NAD+ oxidoreductase
Substrate: L-lactate
Coenzyme: NAD+ (nicotinamide adenine dinucleotide)
Recommended Name: LD
LDH (older term)

Enzyme Classification and Nomenclature

● Classification of Frequently Quantitated Enzymes (Refer to table)
 ● Energy required to raise
all molecules in 1 mole of
a compound at a certain
temperature to the
transition state at the peak
of the energy barrier.
● General Relationship:
○ E: enzyme
○ S: substrate:
○ ES: enzyme -
substrate complex
○ P: product
Illustration explanation:
● X axis: Reaction forming
products from the reactants.
● Y axis: Energy
● In an uncatalyzed reaction, the energy barrier is very high.
○ We will be needing a high activation energy to allow product
formation.
● When you use an enzyme to catalyze or hasten the reaction
(catalyzed reaction), its effect would be to lower energy barrier.
○ We will be needing lower activation energy to allow product
formation.

Types of Specificity
● Enzymes have different specificities in terms of their binding
capacity to their corresponding substrates.
1. Absolute specificity
● Occurs when an enzyme combines with only one substrate
and catalyzes only one corresponding reaction.
2. Group specificity
● When an enzyme combines with all the substrates containing
a particular chemical group. For instance:
○ Esterases enhancing or catalyzing ester compounds only.
3. Bond specificity
● Depends on the type of bond that is present in the compound.
4. Stereoisomeric specificity
Enzyme Kinetics
● Enzymes that predominantly combine with only one optical
● It describes the catalytic mechanism of enzymes to convert the
isomer of a certain compound.
reactants to products.
Factors that influence enzymatic reactions
● Reactions occur spontaneously when the available energy for the
● pH:7.0-8.0
reactants are higher than the products as the reaction proceeds
○ Changes in pH may actually denature an enzyme or influence
toward the lower energy.
its ionic state may result in structural changes or a change in
● Activation energy
the charge of an amino acid residue in the active site
○ Excess energy of the reactants or the energy required to raise
○ It’s important that enzyme operates within a specific pH range
the all mole in one mole of compound in a certain temperature
and maximally at a specific pH
to the transition state at the peak of the energy barrier.
○ Physiologic enzymatic reactions occur in the pH range of
○ Simply, the excess energy needed to overcome the energy
7.0-8.0
barrier and allow the formation of the product.
○ Some enzymes may react at an alkaline pH like alkaline
phosphatase. Some at acidic pH like acid phosphatase.
Ways to allow product formation:
● Temperature: 10 increase, reaction doubles
● Increase the temperature and increase intermolecular collisions
○ Increasing the temperature increases the rate of a chemical
○ However, this does not normally occur in the physiological
reaction by increasing the movement of molecules and the rate
conditions.
at which intermolecular collisions occur
○ We cannot increase temperature in our body nor we can ■ This is the case of enzymatic reactions until the
increase intermolecular collisions.
temperature is high enough to denature the protein
● Provide more energy but this will mean the need to produce more
composition
energy as well.
○ Utilizing extremely low temperature also influences the reaction
● Use enzymes
rate
○ The best possible way to allow product formation.
■ Reversibly inactivates the the enzyme
○ It catalyzes or hastens physiologic reactions by lowering the
■ Utilized in the laboratory in cases when you cannot
activation energy level that the reactants must reach for the
immediately analyze the sample
reaction to occur. → We usually place the specimen in refrigerator or
freeze it to present activity loss until analysis
● Cofactors
○ nonprotein entities(?) that bind to an enzyme before a reaction
occurs
Activation Energy
 ○ Could be activators( inorganic cofactors) or coenzymes
(organic cofactors)
○ May be essential for the reaction to enhance the reaction rate
in proportion with the concentration to the point at which the
excess activator begins to inhibit the reaction
○ Activators function by alternating the spatial configuration of the
enzyme for proper substrate binding, linking the substrate to
the enzyme/coenzyme, or undergoing oxidation/reduction
● Substrate concentration
● Enzyme concentration
● Inhibitors (competitive, noncompetitive, and uncompetitive)

Substrate concentration
● Michaelis Menten
○ S readily binds to free E (enzyme) at a low [S] (substrate
concentration)
○ Addition of S increases the reaction (first order kinetics)
■ Since enzyme concentration is more than substrate
concentration, adding substrate increases the reaction
→ Because the reaction rate is directly proportional to
the substrate concentration, it is called first order
kinetics
■ Reaction depends on substrate
○ [S] is high to saturate E and only free E upon product formation
reacts with S (zero order kinetics)
■ With the addition of substrate, a point will come when
enzymes are saturated and the reaction rate or velocity
reaches maximum
■ The reaction will only happen once a product is formed
and releases the used enzyme
→ The used enzyme being ready to combine with a new Competitive Inhibition
substrate ● Same Vmax; Increased Km
■ Reaction depends on availability of the enzyme (substrate is also increased)
■ Increasing the substrate also increases the reaction ● Inhibitor may bind to the active
velocity but will reach a plateau once enzyme is saturated site and compete with the
by the substrate → reaching maximum velocity substrate for the active site
● Reversible by adding more
Michaelis Menten Constant (Km) substrate so that the substrate
● Relationship between the velocity of an enzymatic reaction and will more likely bind to the active
substrate concentration site than the inhibitor
○ Km is concentration at which the enzyme yields half the
possible maximum velocity thus it is the amount of substrate
needed for a particular enzymatic reaction Noncompetitive Inhibition
● Amount of substrate needed for a particular enzymatic reaction ● Vmax not achieved; Same Km
● ￪ [S], velocity of reaction depends on [E] (substrate concentration remains the
○ As long as the substrate concentration exceeds the enzyme same)
concentration (zero order kinetics), the velocity of the reaction ● The inhibitor binds to a site other
is proportional to the enzyme concentration than the active site
● ￪ [E], the faster the reaction ● This could be reversible if the
○ Because more enzyme is present to bind with the substrate inhibitor will not change any part of
● Theoretically, Vmax and Km can be determined from the plot the enzyme involved in catalytic
○ However, Vmax is difficult to determine from the hyperbolic plot activity
and often not actually achieved in enzymatic reactions because ● Could also be irreversible if the
enzymes may not function optimally in the presence of inhibitor destroys a part of the enzyme involved in catalytic activity
excessive substrate ● Because inhibitor binds with the enzyme, addition of a substrate
■ A more accurate representation of Vmax and Km may be does not reverse the reaction
made through a Line-Weaver Burk plot, a double
reciprocal plot of the Michaelis Menten constant which Uncompetitive Inhibition
yields a straight line ● Vmax not achieved; Decreased Km
(need to decrease substrate to
avoid more ES complex and thus
avoiding more inhibition)
● Inhibitor binds to enzyme-substrate
complex
● Adding more substrate will actually
worsen the reaction and there would
be no product formation
 Measurement of Enzyme Activity
● Catalytic activity not concentration
○ Since enzymes exist in little or minute concentration, we are
not measuring their concentration but rather, their activities
■ Measured activity is then related to their concentration
● What is measured?
○ Increased in product formation
○ Decrease in substrate concentration
○ Decrease in coenzyme concentration
○ An increase in concentration of an altered coenzyme
● Done at zero order kinetics
○ Because reaction rate is dependent upon the enzyme and we
are after the activity of the enzyme
● Note: monitor pH, no inhibitors, temperature

Methods of Measuring Enzyme Activity

● Fixed time or Endpoint measurement
○ Involves combining the reactants allowing the reaction to
proceed and stop by adding a weak acid
○ After stopping the reaction, a single absorbance measurement
at a specific time is done
○ Since it involves one measurement, we assume the reaction is
the same or linear all throughout
● Continuous monitoring or kinetic assay
○ Requires multiple measurement and we observe the
absorbance change at specific time intervals
○ More reliable because we do not assume linearity is present

Calculation of Enzyme Activity

● Activity units: Bodansky and King units
○ Historically, specific method developers frequently established
their own units for reporting results and often name the units
after themselves
○ To standardize the system of reporting quantitative results, the
enzyme commission of the International Union of Biochemistry
defined the international unit or IU as the amount of enzyme
that will catalyze the reaction of 1 micromol if substrate per
minute under specified condition of temperature, pH, substrates
and activators
○ Although the unit may be standard, but the reference value will
still be laboratory specific depending on the laboratory,
reagents utilized, and some other conditions
● IU
○ Amount of enzyme that will catalyze the reaction of 1 umol of
substrate per minute under specified condition
● Enzyme activity recognized by the International System of Units:
katal (mol/s)
● Enzyme concentration: IU/L or kat/L
○ 1.0 UI = 17 nkat

Enzymes as reagents
● Glucose (glucose oxidase and hexokinase), cholesterol (cholesterol
oxidase and cholesterol esterase), uric acid (uricase), TG
● Other enzymes
● Competitive and noncompetitive immunoassays (HRP-horseradish
peroxidase, ALP, G-6-PD-glucose 6 phosphate dehydrogenase,
B-galactosidase)
○ As indicators