Anatomy

Biochemistry
Enzymes and Enzyme Kinetics
Dr. Floro B. Madarcos

1
23 Aug 2016

Dizon, Domingo, Ebreo, Ebuengan, Elarmo

TERMS

Content Outline
I.

II.
III.
IV.
V.
VI.
VII.

VIII.
IX.
X.
XI.

Definition of Terms
A. Enzymes
B. Apoenzymes
C. Cofactors
D. Holoenzyme
E. Metalloenzyme
F. Regulatory Enzyme
G. Active Site of Enzyme
H. Allosteric Site of Enzyme
I. Substrate
Six Major Classes of Enzymes
Cofactors of Enzymes
Characteristics of Enzymes
Models of Enzyme-Substrate Complex
Kinetics of Enzyme Catalyzed Reactions
Illustration of Enzyme Kinetics Using Plots
and Equations
A. Michaelis-Menten Equation
B. Michaelis-Menten Saturation Curve
C. Significance of Michaelis-Menten
Constant
D. KM and the Physiological Utilization of
Glucose
Enzyme Inhibition and Is Effect on
Reaction Velocity
Regulation of Enzymatic Activity
Factors Affecting Enzymatic Activity
Uses and Clinical Application of Enzymes
LEARNING OBJECTIVES

At the end of the lecture, the student should be able to:

1. Define the following:
a. Enzymes
b. Apoenzyme
c. Coenzyme
d. Holoenzyme
e. Metalloenzyme
f.
Regulatory enzyme
g. Active site of the enzyme
h. Allosteric site
i.
Substrate
2. Discuss the characteristics of enzymes
3. Enumerate the six major classes of enzymes
4. Explain the models of enzyme-substrate complex
5. Explain enzyme kinetics
a. Factors that affect enzyme activity or reaction
velocity
b. Ways of expressing enzyme activity
6. Discuss the operation and plots used to illustrate enzyme
kinetics
a. Michaelis-Menten kinetics
b. Lineweaver-Burke double reciprocal plot
c. Kinetic order of reactions
7. Discuss enzyme inhibition and its effect on reaction
velocity
a. Reversible
b. Irreversible
8. Discuss the different ways of regulating enzyme activity
9. Explain the factors affecting enzyme activity
10. Elucidate uses and clinical application of enzymes

1.

2.
3.

4.
5.
6.

7.

8.

9.

ENZYMES specialized protein catalysts that accelerate

chemical reactions (106 to 1020 times faster) but are not
consumed during the reaction and are regenerated for
reuse.

The compartmentalization of enzymes in different

cell parts means specific metabolic pathways are
present only in a specific part of the cell. This
allows pathways to utilize the enzymes.

Do not change or alter the equilibrium position of

the chemical reaction
APOENZYMES the protein part of the enzyme (without
cofactors or prosthetic groups); catalytically inactive
COFACTORS small, soluble non-protein component
required for apoenzyme activity; reversibly bound to
enzyme and participates in overall catalytic activity; helps
bind substrate and catalyze its chemical transformation
into product

Coenzymes could be metals or complex organic

molecules

Prosthetic group molecule permanently attached

to the apoenzyme; most commonly metal ions

Metal ions inorganic (K+, Fe2+, Mg2+, Zn2+)

HOLOENZYME complete enzyme complex; apoenzyme
+ cofactor; catalytically active form of enzyme
METALLOENZYME enzymes that require a metal in
their composition; bind to and retain metal atoms under all
conditions due to high affinity
REGULATORY ENZYME catalyzes the ratelimiting/committed step of a metabolic pathway; its activity
is affected by several factors (positive & negative) so that
an increase/decrease in the enzymes activity rate
changes the rate of the entire metabolic pathway
ACTIVE SITE OF ENZYME site where substrate binds
for a catalytic reaction to occur; located in clefts or
crevices

The surface of the active site is lined with

functional groups that participate directly in the
reaction
a) Amino acid residues could be
polar/non-polar depending on the
substrate
b) Cofactors coenzymes & metal ions
ALLOSTERIC SITE OF ENZYME site where small
molecules (effectors/regulators) bind, that is physically
separate from the enzymes catalytic site; allosteric
modifiers/effectors bind to this site and may cause a
conformational change in the enzyme; cause the active
site to be more active or less active
SUBSTRATE molecule acted upon by the enzyme to
form a product

Enzymes and Enzyme Kinetics

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CHARACTERISTICS OF ENZYMES
1. They are not changed by the reactions they are
catalyzed. However, they may be temporarily modified
over the course of the reaction
2. Do not change or alter the equilibrium position of the
reaction;
a. They do not push a reaction that is
thermodynamically unfavorable (non-spontaneous or
+G)
b. They do not change the free energy of the reaction
(G w/ catalyst = G w/o catalyst).
3. They increase reaction rates by decreasing the required
activation energy for the reaction to proceed by lowering
the energy of the transition state.
a. Transition state unstable complex formed from
reactants; higher free energy than substrate and
product
b. Activation energy (Ea) - energy needed to form
transition state; difference in free energy between the
transition state and substrate (reactant)
c. Some enzymes lower activation energy by
increasing the stability of the transition state

Coenzyme

Acetyl CoA
carboxylase

Acetyl groups

Pantothenic
acid & other
com-pounds

Nicotinamide
Adenine
Dinucleotide
(NAD)

Lactate
dehydrogenase,
Other
dehydrogenases

Hydride ion (H
atom with 2
electrons)

Nicotinic
acid
(Niacin)
(Vit B3)

Biocytin

Pyruvate
carboxylase,
acetyl CoA
carboxylase,
Propionyl CoA
carboxylase

CO2

Biotin

Thiamine
Pyrophosphate
(TPP)

Pyruvate
dehydrogenase,
a-ketoglutarate
dehydrogenase,
a-ketoacid
dehydrogenase
Succinate
dehydrogenase,
pyruvate
dehydrogenase,
nitric oxide
synthase
Glycogen
phosphorylase,
ALA synthase,
histidine
decarboxylase,
alanine amino
transferase

Aldehydes

Thiamine
(Vit. B1)

Electrons

Riboflavin
(Vit B2)

Amino groups
(NH3)

Pyridoxine
(Vit B6)

Pyruvate
dehydrogenase,
a-ketohlutarate
dehydrogenase
Methylmalonyl
mutase
Thymidylate
synthase

Electrons
& Acyl groups

Not required
in diet

H atoms and
alkyl groups
One carbon
groups

Cobalamin
(Vit B12)
Folic acid

Pyridoxal
Phosphate
(PLP)

Lipoate

As proteins, enzymes are made up of chains of amino

acids joined by a peptide bond function is dependent on
its amino acid sequence.

5-deoxy
cobalamin
Tetrahydro
Folate
(FH4; TH4)

COFACTORS (HELPERS) OF ENZYMES

Cofactors are molecules attached to the apoenzyme to

make it catalytically active. (cofactor + apoenzyme =
holoenzyme)
They are either coenzymes (usually vitamin derivatives),
prosthetic groups, or metal ions (metalloenzymes). They
are temporary carriers of specific functional groups during
metabolism.
They have very little activity and specificity in the absence
of the enzyme.

Vitamin
Precursor

Coenzyme A
(CoASH)

Flavin
Adenine
Dinucleotide
(FAD)

Figure 1. Reaction Coordinate Diagram

4. They are highly specific for the reactants/substrates
they act on.

COENZYMES
Apoenzyme
Group
Transferred

Table 1. Summary of coenzymes

COENZYME A
It is derived from pantothenic acid and is a cofactor of
several enzymes like acetyl CoA carboxylase
It takes part in reactions of the Citric Acid Cycle, Fatty Acid
synthesis and oxidation, acylations, and cholesterol
synthesis
It contains an active sulfhydryl group that forms thioesters
with acyl groups (e.g. acetyl, succinyl, or fatty acyl)
Used in -Ketoglutarate dehydrogenase complex in Krebs
cycle, as well as in pyruvate dehydrogenase complex

Enzymes and Enzyme Kinetics

NICOTINAMIDE ADENINE DINUCLEOTIDE (NAD)

It contains a nicotinamide ring from niacin
Its AMP provides additional binding interactions that
induce conformational changes in the enzyme.
It serves as an acceptor in lactate pyruvate reaction
BIOTIN (BIOCYTIN)
It carries CO2 as a bicarbonate in:
o Conversion of Pyruvate to Oxaloacetate in
gluconeogenesis
o Conversion of Acetyl CoA to Malonyl CoA in de novo
synthesis of fatty acids
THIAMINE PYROPHOSPHATE (TPP)
It has a reactive C atom that carries the aldehyde groups.
It is used in decarboxylations in the Krebs Cycle/TCA
o Pyruvate acetyl CoA
o Isocitrate (C6) -Ketoglutarate (C5)
o -Ketoglutarate Succinyl CoA (C4)
- transketolase reaction of PPP
- catabolism of branched-chain amino acids
FLAVINE ADENINE DINUCLEOTIDE (FAD)
It is involved in redox reactions, as an H acceptor
a. Accepts H atom when succinate fumarate
in TCA
It is reduced to FADH2
It is more easily oxidized than NAD and is best used when
2 H+ are to be removed

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CLASSES OF COENZYMES

CLASS
Activation-Transfer
Coenzymes

EXAMPLES
TPP
Coenzyme A
Biotin
PLP

Oxidation-Reduction
Coenzymes

NAD+
FAD
Vit E
Vit C

Table 2. Examples of classes of coenzymes

COFACTOR
+2

Zn

PYRIDOXAL PHOSPHATE (PLP)

It is derived from Vit. B6 and is involved in carbohydrate,
amino acid, and neurotransmitter synthesis.
It transfers amino acids via the functional group of reactive
aldehydes.
Covalently bonds with the enzyme to form Glucose 1-P in
carbohydrate metabolism
It is the cofactor of ALA synthase in the first step of heme
synthesis. It is also involved in histamine synthesis when
histidine is converted to histamine. It also carries an NH3
group from alanine to -Ketoglutarate pyruvate and
glutamate (enzyme: alanine transaminase)

LIPOATE
Dihydrolipoyl transacetylase uses lipoate and CoA to transfer
an acetyl from the lipoyl to thiol of CoA, which produces acetyl
CoA, which enters the citric acid cycle.
5-DEOXYCOBALAMIN
It functions as a reversible free radical generator in the
hemolytic cleavage of AdoB12s C-Co(III) bond. The C and Co
atoms each acquire one of the electrons that formed the
cleaved electron pair bond. The Co ion fluctuates between its
Co(III) and Co(II) oxidation states.
TETRAHYDROFOLATE
An important cofactor in the synthesis of purine bases,
thymine, choline phospholipids, creatinine, epinephrine, and
DNA methylation

INORGANIC COFACTORS
Metal ions are positively charged, and contribute to the
catalytic process by acting as electron-attracting groups
or electrophiles.
They assist in substrate binding, or stabilize anions in the
reaction.
They can also accept or donate electrons in redox
reactions.

Cu+2
+2

ENZYME
Carbonic anhydrase, alcohol
dehydrogenase,
carboxypeptidases A & B
Cytochrome oxidase

Mn

Arginase, Ribonucleotide
reductase

Mg+2

Hexokinase, Pyruvate kinase,

Glucose 6-phosphatase

Ni

+2

Urease

Mo

Nitrate reductase

Se

Glutathione peroxidase

K+

Propionyl CoA carboxylase

Table 3. Inorganic cofactors

1.
2.

ATP & GLUCOSE

ATP can function as a second substrate, especially in
kinase-catalyzed reactions
In the phosphorylation of glucose to glucose 6-phosphate
(1st reaction of glycolysis) catalyzed by either hexokinase
or glucokinase, the terminal phosphate group of ATP,
having a high free energy of hydrolysis, is transferred to
the acceptor molecule glucose. It is phosphorylated at
carbon 6, forming glucose 6-phosphate.

REGULATORY ENZYMES
Phosphofructokinase I
o Catalyzes the phosphorylation of fructose 6-phosphate
fructose 1, 6-bisphosphate in the presence of ATP or
energy, which is the committed step of the glycolytic
pathway, making phosphofructokinase I the regulatory
enzyme

Enzymes and Enzyme Kinetics

o It is affected by several factors (AMP, ATP, fructose 2, 6bisphosphate, citrate, H+)
Acetyl CoA Carboxylase
o Catalyzes the carboxylation of acetyl CoA malonyl CoA
o It is the rate-limiting enzyme of de novo synthesis of fatty
acids
o It is affected by substrates, products, or coenzymes in the
pathway
HMG CoA Reductase
o Catalyzes the reduction of HMG CoA into mevalonate,
which is the rate-limiting step in cholesterol synthesis.
o Its activity is increased by insulin, T3 (thyroxine, a thyroid
hormone) and glucocorticoids. Its activity is decreased by
cholesterol, bile acids, mevalonate, glucagon, and statins.
Glucose 6-Phosphate Dehydrogenase
o Catalyzes the conversion of glucose 6-phosphate into 6phosphogluconolactone, the rate-limiting step of the
pentose phosphate pathway (PPP)
*the rate-limiting step is slower than other steps. The activity of the
enzyme that catalyzes the rate-limiting step is modulated by certain
factors, and so the modulation of the rate-limiting step is enough to
produce a large effect on the rate of formation of the final product.

ISOENZYMES
Enzymes with different amino acid sequences but catalyze
the same chemical reactions
They act on the same substrates and produce the same
products, but are different in degrees of efficiency
They are products of different genes and can have
different kinetic properties
Different isoenzymes are expressed in specific tissues of
the body.
o Lactate dehydrogenase (LDH), which catalyzes the
reversible conversion of pyruvate to lactate, is a
tetramer consisting of 2 subunits: M (found in skeletal
muscles and liver), and H (found in the heart.)
o LDH has 5 distinct isoenzyme forms, all combinations
of M and H isoenzymes. An increase of H in the blood
indicates tissue damage as in a heart attack.

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TRANSFERASE
Transfers functional groups from donors to acceptors
Kinase transfers phosphate (functional group) from ATP
to an acceptor

Glycosyltransferase formation of glycosidic linkage

o Galactosyl transferase in sphingolipid synthesis
Example:
Alanine transaminase
Hexokinase/Glucokinase in glycolysis

SIX MAJOR CLASSES OF ENZYMES

CLASS
Oxidoreductase

SUBCLASS
Dehydrogenases, Oxidases,
Reductases, Peroxidases,
Catalases, Oxygenases,
Hydroxylases
Transferases
Transaldolase and
Transketolase: acyl, methyl,
glucosyl, and
phosphoryltransferases,
Kinases, Phosphomutases,
Transaminases
Hydrolases
Esterases, Glycosidases,
Peptidases, Phosphatases,
Thiolases, Phospholipases,
Amidases, Deaminases,
Ribonucleases
Lyases
Decarboxylases, Aldolases,
Hydratases, Dehydratases,
Synthases, Lyases
Isomerases
Epimerases, Isomerases,
Mutases, Racemases
Ligases
Synthetases, Carboxylases
Table 4. Major classes of enzymes and their examples
(IUBMB, 1964).

OXIDOREDUCTASE
Catalyzes transfer of electrons and hydrogen atoms;
oxidation-reduction reactions
o Oxidation = Loss of Electrons (LEO)
o Reduction = Gain of Electrons (GER)
o alternatively: Oxidation Is Loss, Reduction is Gain (OIL
RIG)
Example: Lactate dehydrogenase

HYDROLASE
Catalyzes cleavage of chemical bonds by the addition of
H2O, producing two products.
Example: Pyrophosphatase
LYASE
Cleaves C-C, C-O, and C-N bonds through means other
than hydrolysis or oxidation.
Example: Pyruvate decarboxylase
o DOPA decarboxylase in the formation of
dopamine
o Histidine decarboxylase in the formation of
histamine from histidine
o Aldolase in glycolysis
Synthase catalyzes a physiologically important reaction
favoring the formation of a carbon to carbon (C-C) bond
o An example is citrate synthase catalyzing the
formation of citrate from Acetyl CoA and
oxaloacetate in the Krebs cycle.
o Another example: cystathionine synthase
catalyzing the formation of cystathione from
homocysteine and serine.
Hydratase adds H2O to a substrate
o Hydratase in the beta-oxidation of fatty acids
ISOMERASE
Transfers functional groups or double bonds within the
same molecule.
Example: Phosphohexoisomerase catalyzing the
formation of aldose to ketose and vice versa (glycolysis)
o Phosphoglycerate mutase catalyzing the
formation of 3-Phosphoglycerate to 1Phosphoglycerate and vice versa (glycolysis)
o Epimerase catalyzing the formation of D-Xylulose
5-phosphate to D-Ribulose 5-phosphate and vice
versa.
LIGASE
Catalyzes the linking of substrates in the presence of ATP
Example: Pyruvate carboxylase (gluconeogenesis)

Enzymes and Enzyme Kinetics

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MODELS OF ENZYME-SUBSTRATE COMPLEX

LOCK AND KEY MODEL

Substrate binds to an enzyme which has an active
site whose shape complements the substrate (like a
lock and a key)
Does not take into consideration 3D flexibility of
proteins
Rigid

Enzyme E binds to the substrate S

(substrate binding) to form an enzymesubstrate complex ES (sometimes called the
Michaelis complex), with rate K1 (K1 = rate
constant for the formation of ES)

Equation 1. Equilibrium Equation Demonstrating

Formation of ES Complex
o

The ES complex has 2 possible fates

It can dissociate to E and S, with a

rate constant of K -1

It can proceed to form product P

(catalytic step), with a rate constant
of K2 (K2 = the rate constant for the
conversion of P from the enzyme
E), K-2 represents the regeneration
of ES from E and P

Figure 2. Lock and Key Model

INDUCED FIT MODEL

As the substrate approaches the enzyme, the
enzymes structure changes to accommodate the
shape of the substrate.
The change in the enzymes conformation promotes
the reaction by repositioning functional groups.
Not Rigid

Equation 2. Equilibrium Equation Demonstrating

Formation of E and P

Equation 3. Overall Reaction Summarized

Figure 3. Induced Fit Model

KINETICS OF ENZYME-CATALYZED REACTIONS

Kinetics: the study of reaction rates (velocities)
- In enzymes: the rate of catalysis (V) vs substrate
concentration [S]

Refers to the quantitative measurement of how an

enzyme works

In typical enzyme-catalyzed reactions, the reactant

and product concentrations are usually hundreds or
thousands times greater than the enzyme
concentration

Consequently, each enzyme molecule catalyzes the

conversion to product of many substrates

Take note that in all catalysts, the enzyme is

regenerated at the end of the reaction
It is useful for measuring:
o Concentration of an enzyme in a mixture (by
its catalytic activity)
o Purity (specific activity)
o Catalytic efficiency and/or specificity for
different substrates
o Comparison of different forms of the same
enzyme in different tissues or organisms
o Effects of inhibitors

ILLUSTRATION OF ENZYME KINETICS

USING PLOTS AND EQUATIONS
a.
b.

MICHAELIS-MENTEN EQUATION
It is a quantitative description of kinetics of enzymecatalyzed reactions.
It describes the relationship between V0 (the initial
velocity of a reaction), [S], Vmax, and KM.
i. Vmax of an enzyme refers to the maximal velocity that
can be achieved at an infinite concentration of a
substrate.

Enzymes and Enzyme Kinetics

ii. KM of the enzyme for a substrate refers to the
concentration of the substrate required to reach of
the Vmax (half-maximal velocity); KM affinity

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dissociates from the enzyme, so that the enzyme

may again combine with more substrates.

Equation 4. Michaelis-Menten Equation

Vo = Velocity at any time (moles/time)
Vmax = Maximal velocity (or reaction rate)
KM = Michaelis constant for the particular
enzyme under investigation (Molarity)
= (K-1 + K2)/K1
[S] = Substrate concentration (Molarity)

MICHAELIS-MENTEN SATURATION CURVE

It is a graphical representation of the MichaelisMenten equation.
It is used to explain the kinetics of enzyme-catalyzed
reactions; a basic model for non-allosteric enzymes. It
describes how reaction velocity varies with substrate
concentration.
Points A, B, and C mark the key features of the curve.
For many enzymes, the rate of catalysis (V0) is
defined as the number of moles product formed/unit
time, varies with substrate concentration.
For a given quantity of enzyme, reaction velocity (V0)
linearly increases as substrate concentration
increases, and begins to level off and approaches a
maximum at higher substrate concentrations (Vmax).
o The graph shows that the maximum is
approached asymptotically, hence a
hyperbolic curve or rectangular
hyperbola.
At substrate concentrations near point A ([S] < KM),
reaction velocity appears to be proportional to the
substrate concentration.
o *The enzyme is still dependent on [S].
Reaction is therefore said to be first order.
At point B, exactly half of the enzyme molecules are
in an ES complex at any instant, and the reaction
velocity rate is exactly of Vmax ([S] = Vmax/2).
o Thus the Michaelis-Menten constant (KM) is
substrate concentration [S] wherein velocity
is Vmax/2 (KM is the substrate concentration
at Vmax/2).
At infinitely high substrate concentration (near point
C) when the [S] is >> KM, V0 = Vmax, the reaction
velocity rate is maximal.
o The reaction is said to be in zero order
because almost all of the enzymes are
bound to the substrates. The rate is
therefore said to be independent of [S].
o Any further increase in [S] will no longer
affect the reaction velocity because almost
all of the enzymes are saturated with the
substrate.
o No free enzymes will be available for forming
the ES complex.
Under these saturating instances, velocity depends
solely on the rapidity within which a product

Figure 4. Michaelis-Menten Saturation Curve

A = [S] < KM
B = [S] = Vmax/2
C = [S] > KM
= V0 is maximal (Vmax)

Figure 5. Representation of an Enzyme in the Presence of

a Substrate
KM AND THE PHYSIOLOGICAL UTILIZATION OF GLUCOSE

The significance of KM is best understood with a

reaction involving glucose oxidation.

Hexokinase (found in extrahepatic cells, including

RBCs) has a KM value of 0.1 mM for glucose,
whereas glucokinase (found in the liver, and
pancreatic -cells (an isoenzyme of hexokinase)) has
a KM value of 5 mM. Thus, Glucokinase has less
affinity for glucose compared to Hexokinase.

When blood glucose is low (ex: occurs in fasting state

or during starvation), hexokinase is used to
phosphorylate glucose into glucose-6-phosphate. The

Enzymes and Enzyme Kinetics

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brain is therefore assured of its energy needs even in

times of glucose depletion.
Glucokinase, on the other hand is only active when
blood glucose is high (ex: immediately after a meal
because glucokinase affinity for glucose is high only if
there is elevated blood glucose)
The high KM of hepatic glucokinase thus promotes the
storage of glucose as liver glycogen or as fat in
adipose tissues (only when there is an excess in
glucose supply).

Figure 8. Graphical Representation of the LineweaverBurke Double Reciprocal Equation

Figure 6. KM and Physiological Utilization of Glucose

The reciprocal of the reaction velocity (1/V) is plotted

on the y-axis and (1/[S]) is plotted on the x-axis
The slope of the line is KM/Vmax
The y intercept is 1/Vmax
The x intercept is -1/KM

ENZYME INHIBITION AND ITS EFFECT ON REACTION

VELOCITY
ENZYME INHIBITION
Inhibitor of an enzyme a compound that binds to the enzyme
resulting in a decreased reaction velocity
Enzyme inhibition is not part of the normal reaction.
Reversible Inhibition
Non-covalently bound to the enzyme and can dissociate at a
significant rate; when inhibitor concentration drops, enzyme
activity is returned back to normal

Figure 7. M-M Saturation Curve of Hexokinase and

Glucokinase

Competitive Inhibition

Inhibitor binds specifically at active or catalytic site,

where it competes with substrate for binding

Inhibitor is a close structural analog of the substrate

Inhibition is reversed by increasing substrate

concentration

LINEWEAVER-BURKE DOUBLE RECIPROCAL PLOT

It linearizes (hence the straight line instead of the

hyperbolic curve) the Michaelis-Menten equation. It is
therefore a more precise way to measure the Vmax and
KM of an enzyme.

It is easier to draw the best straight line through a set

of points than to estimate the best fit of points to a
curve.

It is useful in the analysis of enzyme inhibition and

distinguishing between various enzymatic reaction
mechanisms.

Figure 9. Lineweaver-Burke Plot for Competitive Inhibition.

Vmax unchanged; Km increased to reach a given velocity
Noncompetitive Inhibition

Enzymes and Enzyme Kinetics

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Inhibitor does not compete with a substrate for its

binding site
Inhibitor is a structural analog of substrate B and is a
noncompetitive inhibitor of substrate A
Inhibition is not reversed by the increase of substrate
A because inhibitor binds to substrate Bs binding site

Figure 12. Representation of Reversible Inhibitions. From

left to right: Competitive, Normal, Noncompetitive
Irreversible Inhibition
Inhibition by the modification that occurs at the active site of
the enzyme and the inhibitor. Inhibitor covalently binds to
active enzyme.
Figure 10. Lineweaver-Burke Plot for Noncompetitive
Inhibition.
Vmax decreases proportionately to inhibitor concentration; Km
unchanged (since substrate A can still bind to enzyme)
Uncompetitive Inhibition

Inhibitor does not compete with a substrate for its

binding site

Inhibitor binds to non-catalytic site of enzymesubstrate complex

Substrate binding modifies enzyme structure, making

inhibitor-binding site available

Inhibition cannot be reversed by increasing substrate

concentration

Table 5. Representative Drugs That Inhibit Specific

Enzymes.
Drug
Enzyme Target
Disease
Statin
HMG-CoA reductase
Hypercholesterolemia
Viagra
Phosphodiesterase
Erectile dysfunction
COX-2
COX-2
Inflammatory
inhibitors
conditions
Antabuse
Aldehyde
Alcoholism
dehydrogenase
SThymidylate
Anti-cancer drug
Fluorouracil
synthetase
Allopurinol
Xanthine oxidase
Hyperuricemima
Amrubicin
Topoisomerase II
CA chemotherapy
Captopril
Angiotensin-converting Hypertension
enzyme
Celebrex
Cyclooxygenase-2
Arthritis
Digitoxin
Na-K-ATPase pump
Heart protein
Agenerase
HIV protease
HIV

REGULATION OF ENZYMATIC ACTIVITY

Figure 11. Lineweaver-Burke Plot for Uncompetitive

Inhibition.
Vmax and Km decreases (hence the production of parallel
lines in uninhibited and inhibited reaction)

Achieved through two general mechanisms:

1. Control of Enzyme Quantity

Altering the rate of enzyme synthesis and

degradation

Induction

Repression
2. Control of Enzyme Activity/Catalytic Efficiency

Feedback Inhibition

Allosteric Modification

Covalent Modification

Zymogen Activation

Induction or Repression of Enzyme Synthesis

Protein-Protein Interaction
o Feedback Inhibition
end product of pathway inhibits the first
enzyme of that pathway
product inhibiting its own synthesis
occurs since synthesis requires a lot of
ATP which would diminish the ATP

Enzymes and Enzyme Kinetics

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reserve for other metabolic processes

(futile cycle: energy death)
e.g. HMG CoA reductase

Figure 14. Graphical Representation of Dephosphorylation

resulting in Inactivation

Figure 12. Graphical Representation of Feedback

Inhibition
o
-

Allosteric Modification
binding of modulator (maybe the
substrate itself or other metabolites) to
allosteric/regulatory site resulting to
change in conformation of regulatory
enzyme, thus changing the activity of
the active site
modulator maybe stimulatory (positive
allosteric molecule) or inhibitory
(negative allosteric molecule)
*Stimulatory: binding of modulator to allosteric site
INCREASES BINDING in ACTIVE SITE
*Inhibitory: binding of modulator to allosteric site
CHANGES CONFORMATION of ACTIVE SITE

ENZYMES

Low
activity

Acetyl CoA Carboxylase

EP

Glycogen synthase

EP

Pyruvate dehydrogenase

EP

HMG CoA reductase

EP

Glycogen phosphorylase

EP

Citrate lyase

EP

Phosphorylase b kinase

EP

HMG CoA reductase kinase

EP

E = Dephosphorylated
EP = Phosphoenzyme
Table 6. Summary of Mammalian Enzymes and Effect of
Covalent Modification on Catalytic Activity
o
-

Figure 13. Steps in Allosteric Modification

o
-

Covalent Modification
Phosphorylation or dephosporylation of
amino acid residues to activate or
inactivate the enzyme

High
activity

Zymogen Activation
Enzyme is initially synthesized as an
inactive precursor (zymogen); cleavage
of zymogen results in its activation
Ex: Blood Coagulation

Figure 15. Graphical Representation of Fibrin

o

Induction or Repression of Enzyme

Synthesis
Induction increase in the rate of enzyme
synthesis by substances called inducers

Enzymes and Enzyme Kinetics

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Constitutive Enzymes concentration of

these enzymes do not depend on inducers

Inducible Enzymes concentration of these

enzymes depends on the presence of
inducers

Ex: Induction of lactase enzyme in bacteria

growth
Repression decrease in the rate of enzyme
synthesis by substances called repressors which
are low molecular weight substances that decrease
enzyme synthesis at the level of gene expression;
repressors are usually end products of biosynthetic
reaction so this is sometimes called feedback
regulation (BUT NOT THE SAME AS FEEDBACK
INHIBITION!).

Ex: Dietary cholesterol decreases rate of

synthesis of HMG-CoA reductase which is a
key enzyme in cholesterol biosynthesis

Protein-Protein Interaction
o Enzymes formed from many
protein subunits may be present in
its inactive form due to its
interaction with its subunits.
Activation occurs following its
separation form its regulatory
subunits

FACTORS AFFECTING ENZYME ACTIVITY

TEMPERATURE

Figure 17. Temperature vs. Reaction Velocity

The increase in velocity before the optimum

temperature is due to increased kinetic or vibrational

Beyond optimum temperature or with further increase

in temperature results to decrease in velocity due to
denaturation of the enzyme, specifically destruction of
the secondary and tertiary structures of the enzyme.
Optimum temperature is the temperature at which the
rate of the reaction is maximal/highest/fastest.
Graph: Slightly skewed to the right
A 10C rise in temperature will increase the activity of
most enzymes by 50 to 100%.
For most human enzymes, optimum temperature is
between 45-55 C.
PH

Figure 16. Graphical Representation of Activation of

Protein Kinase A Enzyme
Protein kinase A enzyme is composed of 2 Regulatory and 2
Catalytic subunits making it inactive. cAMP or cyclic adenosine
monophosphate binds to the 2 Regulatory subunits, releasing
the 2 catalytic subunits thereby activating the enzyme.

Figure 18. PH vs. Reaction Velocity

Velocity of an enzyme-catalyzed reaction increases

with increase in pH until the optimum pH is reached

Before the optimum pH, there is an increasing velocity

of an enzyme-catalyzed reaction due to:
a. Ionization of the specific functional groups of
the amino acids that line the active or
catalytic site of the enzyme (or in the
substrate).
b. General formation of hydrogen bonds
important in the overall conformation of the
enzyme.

Enzymes and Enzyme Kinetics

Optimum pH refers to the pH at which the velocity of

the reaction is highest; the enzyme is in its most
active form.
Beyond optimum pH, there is decreasing velocity due
+
to the deprotonation of an amino terminal NH3

Graph: bell-shaped curve

SUBSTRATE CONCENTRATION

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USES AND CLINICAL APPLICATION OF ENZYMES
Enzymes in Clinical Diagnosis

Under normal physiologic conditions in a healthy

individual, certain intracellular enzymes are released
into the blood during normal cell turnover.

However, in the presence of cell necrosis due to

disease or trauma (heart, liver, skeletal muscles and
other tissues), these enzymes become elevated
hence certain enzymes are markers of diseases or
some enzymes become diagnostic tools for
diseases of certain tissues
CARDIAC ENZYMES AS MARKERS FOR MYOCARDIAL
INFARCTION

Figure 19. Substrate Concentration vs. Reaction Velocity

Velocity of a reaction increases as the substrate

concentration increases until maximal velocity (Vmax)
is reached.

Thereafter, further increases in the substrate

concentration will no longer increase the velocity
because all the enzymes have been "saturated" with
the substrate

Graph: hyperbolic shape of the curve

excessive amounts of substrates after Vmax:

decrease reaction velocity

Explanation: many substrates competing for the

active sites on the enzyme surfaces that they block
the sites prevent any other substrate molecules
from occupying them drop in velocity since the
entire enzyme present is not being used (*They forget
the binding site.)
COFACTORS

Figure 20. Time vs. Rate of Reaction

- +2
+2

The presence of cofactors (like Cl , I , Br , Zn , Mg ,

etc.) increase the rate of an enzyme-catalyzed
reaction.

Cofactor Concentration = Velocity

Figure 21. Appearance of myocardial enzymes over time in

a patient with MI
1. Troponin (Troponin T and Troponin I isoforms)
Regulatory proteins involved in myocardial
contractility, hence are very specific and preferred
markers for detecting myocardial cell injury, as in
myocardial infarction and are therefore not present in
the serum of healthy individuals.
Rises 3-6 hours after injury; peaks in 12-16 hrs; stays
elevated in 5-14 days.
2. Creatine Kinase
Begins to rise 4-6 hours after MI; peak at 24 hrs;
returns to normal in 3-5 days.
Isoenzymes:
a. CK-MM fraction = found in skeletal
muscle
b. CK-MB fraction = found in heart
muscle
c. CK-BB = found in the brain
May be increased in other conditions: physical
exertion, postoperatively, convulsions, delirium
tremens, etc; hence not diagnostic for MI unless the
CK-MB fraction is being assayed: rises in 3-4 hours
after MI; peak 12-14 hrs later and returns to normal in
2 days.
3. Lactate Dehydrogenase
Peak level about 36-40 hrs after MI and thus of
diagnostic value in patients admitted more than 48 hrs
after infarction.
Levels return to normal in 5-14 days.
No longer used to diagnose MI since it is also found in
other tissues like liver, skeletal muscles, red blood
cells and a variety of organs.

Enzymes and Enzyme Kinetics

4. Aspartate Aminotransferase, AST
Rise within 8 hrs after MI; peak at 24-36 hrs; returns
to normal pre-injury level within 3-7 days.
Not diagnostic for MI since the enzyme is also found
in hepatocytes.
ENZYME MARKERS FOR LIVER DISEASE
1. SGPT-ALT (Serum Glutamic Pyruvic TransaminaseAlanine Aminotransferase)
Found predominantly in the hepatocytes, hence
diagnostic of liver pathology.
2. SGOT-AST (Serum Glutamic Oxalocetic TransaminaseAspartate Aminotransferase)
Also found in cardiac muscles, hence may be helpful
in the diagnosis of cardiac injury, as in myocardial
infarction.
When assaying for both ALT and AST, the ratio of the
level of these two enzymes can also be diagnostic.
a. In a liver damage not of viral etiology,
ALT/AST ratio is less than 1.
b. In viral hepatitis, the ratio is greater than 1.
REFERENCES

2019C Trans
Lehningers Principles of Biochemistry, Nelson,
th
D.L., Cox, M.M., 5 ed., pp. 183-220.
Harpers Illustrated Biochemistry, Murray, R. K.,
et. al., 29th ed., pp. 62-82.
Biochemistry with Clinical Correlations, Devlin, M.
th
T., 7 ed., pp. 378-421.
Biochemistry, Lippincotts Illustrated Reviews,
Champe, P.C., Harvey, R.A., 4th ed., pp. 53- 67.
Principles of Biochemistry, Horton, H.R., et al.,
4th ed., pp. 129-156.
Marks Basic Medical Biochemistry: A Clinical
Approach, Lieberman, M., Marks, A.D., 4th ed.,
pp. 112-149.
Biochemistry, Campbell, M.K., Farrell, S.O. 6th
ed.

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