UERMMCI College of Medicine

Subject: Biochemistry
Date:
June 25, 2014
Title:
(LE 1, Lecture 1.4) Enzymes & Enzyme Kinetics
Lecturer: Dr. Floro B. Madarcos
Batch/section: 2018A
Sem/A.Y.: 1st / A.Y. 2014-2015
Transcribers:
Calupitan, C., Candido, S., Canto, G., Capistrano, D., Base, J.
Trans Subject head:
Evangelista, A. (9369390879/alanaevangelista@gmail.com)
I. Outline
A. Definition of Terms
B. Cofactors of Enzymes
C. Six Major Classes of Enzymes
D. Characteristics of Enzymes
E. Models of Enzyme - Substrate Complex
F. Kinetics of Enzyme - Catalyzed Reactions
G. Illustration of Enzyme Kinetics Using Plots &
Equations
H. Inhibition of Enzymatic Activity
I. Regulation of Enzymatic Activity
J. Factors Affecting Enzymatic Activity
K. Clinical Application of Enzymes
II. Objectives
A. Define the following:
- Enzymes, Apoenzyme, Coenzyme, Holoenzyme,
Metalloenzyme, Regulatory enzyme, Active site of the
enzyme, Allosteric site, Substrate
B. Discuss the cofactors of enzymes
C. Enumerate the six major classes of enzymes
D. Discuss the characteristics of enzymes
E. Explain the models of enzyme-substrate complex
F. Explain enzyme kinetics
- Factors that affect enzyme activity or reaction
velocity
- Ways of expressing enzyme activity
G. Discuss the operation and plots used to illustrate
enzyme kinetics
- Michaelis-Menten kinetics
- Lineweaver-Burke Double Reciprocal Plot
- Michaelis constant and its significance
- Kinetic order of reactions
H. Discuss enzyme inhibition and its effect on reaction
velocity
- Reversible
- Irreversible
I. Discuss the different ways of regulating enzyme
activity
J. Explain the factors affecting enzyme activity
K. Elucidate uses and clinical application of enzymes

A. Definition of terms
1. Enzymes specialized protein catalysts that
accelerate chemical reactions in biologic systems to
about 10^5 to 10^20 times faster than uncatalyzed
reactions but are not consumed during the reactions
they catalyse. At the end these are neither used up but
are regenerated, recycled, reutilized
2. Apoenzyme protein part of an enzyme without any
cofactors or prosthetic groups; catalytically inactive;
cannot increase reaction rate by itself
3. Cofactor small organic or inorganic molecules;
either loosely or tightly bound to apoenzyme that
participates in overall catalytic activity; non-protein part
of enzyme
- Coenzymes small organic molecules often
derived from vitamins
- Prosthetic group an organic molecule
permanently attached to the apoenzyme
- Metal ions inorganic molecules
4. Holoenzyme consists of an apoenzyme and a
cofactor; catalytically active form of enzyme; complete
enzyme complex.
5. Metalloenzyme enzymes that require metal in their
composition; has very high affinity in binding and
retaining metal atoms
6. Regulatory enzyme - catalyzes the rate-limiting or
committed step of a metabolic pathway
7. Active site of the enzyme site where the substrate
binds for a catalytic reaction
8. Allosteric site of the enzyme a site where small
molecules (effectors or regulators) bind and may cause
conformational changes in the enzyme; may cause
active site to be more reactive or less reactive.
9. Substrate molecule acted upon by enzyme to form
product
B. Cofactors of Enzymes
1. Coenzymes (Organic Molecules) - Usually (but not
always) derived or synthesized from B- vitamins and act
as transient carriers of specific functional groups; very
little activity and specificity in the absence of the
enzyme.

III. Lecture
Calupitan, C.| Candido, S.| Canto, G.| Capistrano, D.| Base, J.
A.Y. 2014-2015

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Biochemistry1.4 |Enzyme Classification & Kinetics

Table 1. Some Coenzymes and their Properties

Coenzyme

Thiamine
Pyrophosphat
e (TPP)

Flavin
Adenine
Dinucleotide
(FAD)

Nicotinamide
Adenine
Dinucleotide
(NAD)
Pyridoxal
Phosphate
(PLP)

Lipoate

Coenzyme A
(CoA)

Biocytin
5
deoxycobala
min
Tetrahydrofol
alate

Enzyme

Pyruvate
dehydrogenase,
isocitrate
dehydrogenase, aketoglutarate
dehydrogenase,
transketolase, aketoacid
dehydrogenase
Succinate
dehydrogenase, Ketoglutarate
dehydrogenase,
Pyruvate
dehydrogenase,
Nitric oxide
synthase
Lactate
dehydrogenase,
other
dehydrogenases
Glycogen
phosphorylase,
-ALA synthase,
Histidine
decardoxylase,
Alanine
aminotransferase
Pyruvate
dehydrogenase
-Ketoglutarate
dehydrogenase
Acetyl CoA
carboxylase
Pyruvate
carboxylase, Acetyl
CoA carboxylase,
Proprionyl CoA
carboxylase
Methylmalonyl
mutase
Thimdylate
synthase

Chemical
Groups
Transferred

Vitamin
Precursor

Aldehydes

Thiamine
(Vitamin B1)

Electrons

Riboflavin
(Vitamin B2)

Hydride ion
(:H-)

Nicotinic acid
(Niacin; B3)

Amino groups

Pyridoxine
(Vitamin B6)

Electrons and
acyl groups

Not required
in diet

Acyl groups

Pantothenic
acid and other
compounds

CO2 in the
form of
bicarbonate

Biotin

H atoms and
alkyl groups

Vitamin B12

One-carbon
groups

Folic acid

Table 2. Coenzyme Classes and Examples

Class
Examples
Activator-Transfer
TPP, Coenzyme A,
Coenzymes
Biotin, PLP
Oxidation-Reduction
NAD+, FAD, Vitamin E,
Coenzymes
Vitamin C
*Source: Marks Medical Biochemistry; 3rd ed
a. Coenzyme A (CoA or CoASH) a pantothenic acid
derived compound; a cofactor of several enzymes like
acetyl CoA carboxylase; takes part in reactions of CAC,
fatty acid synthesis and oxidation, acylations, and
cholesterol synthesis; active sulfhydryl group binds to

Calupitan, C.| Candido, S.| Canto, G.| Capistrano, D.| Base, J.

A.Y. 2014-2015

acyl groups (ex. acetyl, succinyl, or fatty acyl) to form

thioesters.
i. a coenzyme (together with other coenzymes) of ketoglutarate dehydrogenase complex that oxidatively
decarboxylates -ketoglutarate to succinyl CoA; this is
one of the irreversible and redox reactions of the Citric
Acid Cycle or Krebs Cycle.
ii. also a coenzyme (together with other coenzymes) of
pyruvate dehydrogenase complex which catalyzes the
oxidative decarboxylation of pyruvate to acetyl CoA, the
reaction that links glycolysis with Krebs Cycle.
Pyruvate dehydrogenase - a multienzyme complex,
similar to -ketoglutarate multienzyme complex because
it is made up of 3 enzymes (pyruvate decarboxylase,
dihydrolipoyl transacetylase and dihydrolipoyl
dehydrogenase) and 5 coenzymes (CoA, TPP, lipoate,
FAD, NAD), with each of these enzymes and coenzymes
playing an important role in the overall reaction process.
b. Nicotinamide Adenine Dinucleotide (NAD+,
oxidized form) and Nicotinamide Adenine
Dinucleotide Phosphate (NADP, reduced form)
- coenzymes of dehydrogenases involved in redox
(oxidation-reduction) reactions.
i. difference between NAD+ and NADP+ is the
presence of a phosphate group of the 2' carbon the
adenine ribose.
ii. The lactate dehydrogenase reaction (reoxidation of
lactate to pyruvate) involves NAD+
iii. The functional group of NAD+ is the carbon on the
nicotinamide ring (derived from niacin) opposite the
positively charged nitrogen atom 1 due to its attachment
to a ribose ring.
iv. This positive charge makes it possible for
nicotinamide to accept a negatively charged hydride ion
(colored green; H: an H atom with 2 electrons) from
carbon 2 of lactate.
v. The H+ from lactate (an alcohol, with OH) then
dissociates & converted to H2O, and a keto group (C=O)
in pyruvate is formed.
vi. Finally, NADH dissociates from pyruvate.
vii. Hence NAD+- dependent dehydrogenases catalyze
the oxidation of alcohols to ketones (for example: lactate
to pyruvate).
viii. AMP provides additional binding interactions that
induce conformational changes in the enzyme.
ix. NADP functions by the same mechanism, but it is
usually involved in pathways of reductive synthesis

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Biochemistry1.4 |Enzyme Classification & Kinetics

(synthesis of fatty acids, cholesterol, bile acids and

steroid hormones).
Nicotinamide Adenine Dinucleotide (NAD+)
coenzyme of lactate dehydrogenase reaction; 2 H+ ions
from lactate are transferred to NAD+ and in the process it
gets reduced to NADH+ + H+.
c. Biotin
i. One of the B complex vitamins; a cofactor of such
enzymes as acetyl carboxylase, propionyl carboxylase
and pyruvate carboxylase.
ii. It is a carrier of activated CO2 (as HCO3-) to
compounds in carboxylation reactions.
iii. The reactive N is the site of CO2 attachment.
iv. Biotin as a source of CO2, is a co-factor of pyruvate
carboxylase that catalyzes the carboxylation of pyruvate
into oxaloacetate, the 1st step of gluconeogenesis
v. Biotin is covalently attached to a lysine residue in
the carboxylase enzyme
d. Thiamine Pyrophosphate (TPP)
i. Vit. B1-derived cofactor in energy-yielding
metabolism, especially in carbohydrate and amino acid
metabolism; synthesized in human cells by addition of a
pyrophosphate.
ii. The functional group of TPP is the reactive carbon
which is a carrier of aldehyde groups in metabolism.
iii. In the given example, TPP is a cofactor of several
dehydrogenases (pyruvate dehydrogenase, isocitrate
dehydrogenase and ketoglutarate dehydrogenase) that
catalyze oxidative decarboxylations of the Krebs Cycle.
e. Flavin Adenine Dinucleotide (FAD) a riboflavin
(Vit B3)-derived coenzyme of several dehydrogenases
involved in oxidation-reduction reactions.
e.1. Role of FAD in the Citric Acid Cycle
i.
FAD is a coenzyme of succinate
dehydrogenase
ii.
that catalyzes the conversion of succinate
into fumarate, one of the reactions of CAC.
ii. FAD is a hydrogen acceptor from succinate and in
the process it is reduced to FADH2.
iii. The oxidation of saturated carbon-carbon bonds,
as in the given example (oxidation of succinate to
fumarate) requires an FAD-dependent succinate
dehydrogenase.
e.2. Role of FAD and FMN (Flavin Mononucleotide)
in Nitric Oxide Synthesis

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A.Y. 2014-2015

i. Nitric oxide is a mediator in a broad array of

biologic systems.
ii. It is synthesized from arginine, catalyzed by nitric
oxide synthase, with FAD, FMN, heme and
tetrahydrobiopterin and NAD as coenzymes.
f. Pyridoxal Phosphate (PLP)
i. Vit. B6-derived coenzyme involved in carbohydrate,
amino acid and neurotransmitter synthesis.
ii. The functional group is a reactive aldehyde that
forms a covalent intermediate with amino groups of
amino acids, hence PLP is involved in the transfer of
amino groups.
f.1. Role of PLP in Carbohydrate Metabolism
i. Glycogen phosphorylase, the major enzyme
involved in glycogenolysis (breakdown of glycogen into
glucose in times of less plentiful glucose) contains a
molecule of covalently bound PLP as a coenzyme.
f.2. Role of PLP in Amino Acid Metabolism
PLP as a Coenzyme in Heme Synthesis
i. In the synthesis of heme, glycine and succinyl CoA
condenses to form -aminolevulinic acid; this is the first
step in the biosynthetic pathway.
ii. The reaction is catalyzed by -aminolevulenate
synthase, with PLP as a coenzyme.
PLP as a Coenzyme in Histamine Synthesis
i. Histine is decarboxylated into histamine via
histidine decarboxylase with PLP as a coenzyme.
ii. Histamine is a chemical messenger that
mediates a wide variety of chemical responses, including
allergic and inflammatory reactions, and
neurotransmission in some parts of the brain.
PLP as a Coenzyme in Transamination Reactions
i. Alanine losses its amino group to the acceptor
molecule -ketoglutarate (accepts amino groups),
forming pyruvate and the collector glutamate (collects
amino groups).
ii. The reaction is catalyzed by alanine
aminotransferase, with PLP as a cofactor.
2. Metals or Inorganic Molecules - Metal ions, which
have a positive charge, contribute to the catalytic
process by acting as electophiles (electron-attracting
groups); assist in binding of the substrate, or they
stabilize anions in the reaction; they can also accept or
donate electrons in oxidation-reduction reactions.

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Biochemistry1.4 |Enzyme Classification & Kinetics

Table 3. Metal Ions and their Enzymes

d. Other Metal Cofactors Cu+2, Mn+2, Na+, Fe+2,

Fe , Ca+2, Mo+3
+3

Inorganic
(Metal ions or
iron-sulfur
clusters)

Enzyme

Zn+2

Carbonic anhydrase, Alcohol

dehydrogenase,
Carboxypeptidases A & B

Cu+2

Cytochrome oxidase

Mn+2

Arginase, Ribonucleotide
reductase

Mg+2

Hexokinase, Pyruvate kinase,

Glucose 6- phosphatase

Ni+2

Urease

Mo

Nitrate reductase

Se

Glutathione peroxidase

Mn+2

Superoxide dismutase

K+

Propionyl CoA carboxylase

a. Mg+2
i. A cofactor of hexokinase or glucokinase, an
enzyme that catalyzes the phosphorylation of glucose to
glucose 6-PO4 in the presence of ATP; this is the 1st step
of glycolysis.
ii. Kinases usually require Mg++ as a co-factor.
iii. In the phosphorylation of glucose to glucose 6phosphate, Mg+2 is bound to the phosphates of ATP,
making the phosphate ion more positive and susceptible
for nucleophilic attack, hence kinase can easily catalyze
the transfer of the terminal phosphate group of ATP to
glucose, forming glucose 6-phosphate.

C. Six Major Classes of Enzymes

1. Oxidoreductases transfer of electrons and
hydrogen atoms (oxidation-reduction reactions) from
donors (reductants, hence oxidized) to acceptors
(oxidized, hence reduced)
Oxidation - Loss of electrons; Reduction- Addition of
electrons

2. Transferases transfer functional/ chemical groups

(e.g. C-, N- or P-) from donors to acceptors; utilize 2
substrates to produce 2 products

3. Hydrolases - catalyze cleavage of chemical bonds

by the addition of H2O, producing 2 products

b. K+
i. A cofactor of hexokinase/glucokinase, an enzyme
that catalyzes the phosphorylation of glucose to glucose
6-phosphate, the 1st step of glycolysis.
ii. Also a cofactor of pyruvate kinase, an enzyme that
catalyzes the conversion of phosphoenolpyruvate (PEP)
into pyruvate, the last irreversible reaction of glycolysis.
c. Zn+ - is a cofactor of carbonic anhydrase, an
enzyme that catalyzes the synthesis of H2CO3 from CO2
and H2O; it is bound to the active site of the enzyme.

Calupitan, C.| Candido, S.| Canto, G.| Capistrano, D.| Base, J.

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Biochemistry1.4 |Enzyme Classification & Kinetics

4. Lyases - cleave C-C, C-O or C-N bonds by means

other than hydrolysis or oxidation

Transition state complex of reactants competent

to produce reaction products; higher free energy than
substrate and product

Activation energy - energy needed to form

transition state reactants; difference in free energy
between the transition state and substrate

5. Isomerases - catalyze transfer of functional groups

or double bonds within the same molecule (or catalyze
racemization of optical or geometric isomers)

- An enzymes binds tightly to the transition-state

structure thereby decreasing the activation energy
If it binds to a substrate tightly, reaction needs
more energy to reach transition state
If it binds to a product too tightly, it will be
difficult
to dissociate from the product

6. Ligases - catalyze ligation or joining of 2 substrates

with covalent bonds in a reaction coupled Catalyze
ligation or joining of 2 substrates with covalent bonds in
a reaction coupled

4. Highly specific for the reactants and substrates they

act on
5. Mostly proteins in nature (although small number of
RNA-based biological catalysts called ribozymes have
been identified)
- Chains of amino acids joined together by peptide
bonds; catalytic activity depends on the integrity of their
native protein
- With denaturation, their catalytic activity is lost
D. Characteristics of Enzymes
1. not changed by the reaction they catalyze, although
they may be temporarily changed or modified during the
reaction
2. do not change or alter the equilibrium position of the
reaction; cannot force a reaction that is not energetically
favorable (non-spontaneous); equilibrium would be
attained rapidly in the presence of an enzyme
3. Increase reaction rates by decreasing the activation
energy

Calupitan, C.| Candido, S.| Canto, G.| Capistrano, D.| Base, J.

A.Y. 2014-2015

E. Models of Enzyme - Substrate Complex

Formation of Enzyme Substrate Complex first
step in enzymatic catalysis

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Biochemistry1.4 |Enzyme Classification & Kinetics

1. Lock and Key Model

- The substrate binds to a site whose shape
complements its own, like a key fitting into a rigid lock;
largely historical because it does not take into account
the 3-dimensional flexibility of proteins.

2. Induced Fit Model

- the enzyme undergoes a slight conformational
change ("induced fit") on binding to the substrate;
flexible pocket that approximates the shape of the
substrate active site of the; substrate-binding site is
viewed as a dynamic surface created by the flexible
overall three-dimensional structure of the enzyme.

F. Kinetics of Enzyme - Catalyzed Reactions

- At a constant enzyme concentration, the reaction

velocity increases with increasing substrate
concentration until a maximal velocity is reached.
- At maximal velocity, all the enzymes have been
saturated hence further increases in substrate
concentration will not increase the velocity.
- The fact that an enzyme-catalyzed reaction has a
maximal velocity suggests the formation of a discrete ES
complex.
- Reaction velocity (or rate of chemical reaction) can
be measured in terms of the number of molecules of
reactant(s) that are converted into product(s) in a
specified period of time.

Kinetics of Enzyme Catalyzed Reaction

- quantitative measurement of how an enzyme works
- Enzyme E binds to the substrate S (substrate
binding) to form an enzyme substrate complex ES
(sometimes called Michaelis complex), with rate
constant K1, the rate constant for the formation of ES.
- 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, the rate constant for the
conversion of P from the enzyme E); K-2 represents
the regeneration of ES from E and P.
- In all catalysis, the enzyme is regenerated at the end
of the reaction.
G. Illustration of Enzyme Kinetics Using Plots &
Equations
1. Michaelis-Menten Equation
- Describes the relationship between Vo (the initial
velocity of a reaction), [S], Vmax, and Km.

Slide on Reaction Velocity vs. Substrate

Concentration - graphically demonstrates the existence
of an enzyme-substrate complex.

Calupitan, C.| Candido, S.| Canto, G.| Capistrano, D.| Base, J.

A.Y. 2014-2015

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Biochemistry1.4 |Enzyme Classification & Kinetics

Vo = velocity at any time (mmol/s)

Vmax = maximal velocity (or reaction rate);
maximal velocity that can be achieved at an infinite
concentration of a substrate.
Km = Michaelis constant for the particular
enzyme under investigation; concentration of the
substrate required to reach 1/2 Vmax (or half-maximal
velocity).
= [S] at Vo=Vmax/2
[S] = substrate concentration (mmol)

Km and Physiological Utilization of Glucose

Michaelis-Menten Saturation curve

- Km for hexokinase = 0.1 mM has higher affinity to

glucose; lower amount of glucose needed to attain Vmax;
brain is assured of its energy needs even at times of low
glucose concentration, ie starvation

Points at the graph:

- A: [S] < Km; only a portion of the enzyme is present as
an ES complex
- B: [S] = Vmax/2; half of the enzymes exist as ES
complex, hence velocity is half of Vmax
- C: [S] > Km; reaction velocity rate is maximal

Points A & B - will result to a proportional

increase in velocity; reactions are said to be of
the first order
Point C further increase in substrate
concentration will further increase the velocity
because most of the enzymes are fully saturated
with the substrate; reaction is said to be of the
zero order

- Km for glucokinase = 5 mM has lower affinity to

glucose; active only at high glucose concentration, ie.
after a meal; promotes the storage of glucose as liver
glycogen or as fat in adipose tissues, when glucose is in
excess supply
2. Lineweaver Burke Double Reciprocal Plot
- linearizes (hence straight line instead of the
hyperbolic curve) the Michaelis-Menten Equation; more
precise way to measure Vmax and Km of an enzyme
- easier to draw the best straight line through a set of
points than to estimate the best fit of points to a curve.
The reciprocal of reaction velocity, 1/V, is plotted
on the y axis (ordinate).
The reciprocal of substrate concentration, 1/[S],
is plotted on the x axis (abscissa).
Km/Vmax = slope of the line
1/Vmax = Y intercept
-1/Km = X intercept

Significance of Km
- concentration of substrate at which half of the
active sites of the enzyme are filled up
- inverse measure of the affinity of the substrate for
the enzyme:
The lower the km, the higher is the affinity; Vmax
is reached at relatively low substrate
concentration.
The higher the km, the lower is the affinity; Vmax
can be reached only at high substrate
concentration
Calupitan, C.| Candido, S.| Canto, G.| Capistrano, D.| Base, J.
A.Y. 2014-2015

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Biochemistry1.4 |Enzyme Classification & Kinetics

H. Inhibition of Enzymatic Activity

- inhibition by specific small molecules and ions binding
to the active site, resulting to a decrease in the velocity
of the reaction; ; not part of the normal reaction
1. Reversible non covalent bond of inhibitor to the
enzyme; can dissociate at a significant rate; when
inhibitor concentration drops, enzyme activity is
regenerated
a. Competitive - inhibitor competes with the
substrate for the active site on the enzyme; reversible by
increasing substrate concentration to outcompete with
the inhibitor
b. Non-competitive Inhibitor binds to enzyme but
not at the active or catalytic site; conformational change
in the enzyme; substrate can still bind but with
decreased catalytic power hence, no products formed;
non - reversible w/ increase of substrate concentration
- kinetic effect: Vmax decreased proportionately to
inhibitor concentration; Km unchanged (since
substrate can still bind to the enzyme)
c. Uncompetetive inhibitor binds only to ES
complex at locations other than active site; binding of
substrate and enzyme exposes site available for inhibitor
binding; rare in occurrence; cannot be reversed with
increased substrate concentration
- kinetic effect: Vmax decreased
Km decreased

- Km increases for a given substrate in order to attain

Vmax that were reached in its absence, hence the
differing X intercepts.
Lineweaver-Burke plot for non-competitive inhibition
- Vmax is decreased since a
noncompetitive inhibitor
reduces the concentration of
ES complex that can advance
to reaction products, hence the
differing Y-intercepts
- Km is unchanged since
substrate can still bind to the
enzyme, hence the identical X
intercepts of Lineweaver - Burke plots, with or without
noncompetitive inhibitor.
Lineweaver-Burke plot for uncompetitive inhibition

Uncompetitive inhibitors
decrease both Vmax and
Km, hence the production of
parallel lines in uninhibited
and inhibited reactions.

2. Irreversible inhibition by the modification that

occurs at the active site of the enzyme and the inhibitor

Lineweaver - Burke plot for competitive inhibition

- Vmax unchanged since
increasing substrate
concentration can displace
virtually all competitive
inhibitors bound to the active
site, hence the identical Y-axis
intercepts of Lineweaver Burke plots, with or without
inhibitor.

Calupitan, C.| Candido, S.| Canto, G.| Capistrano, D.| Base, J.

A.Y. 2014-2015

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Biochemistry1.4 |Enzyme Classification & Kinetics

I. Regulation of Enzymatic Activity

1. Feedback Inhibition (or End Product Inhibition)
- products of an enzyme-catalyzed reaction blocks
an earlier enzyme in the sequence, inhibiting the further
synthesis of the products; products in these reactions
serves as a negative feedback mechanism

3. Covalent Modification - involves phosphorylation

(addition of a phosphate group) or dephosphorylation
(removal of a phosphate group) from specific threonine,
tyrosine or serine residues of the enzyme

2. Allosteric (Non covalent) Modification

- An allosteric enzyme binds to an allosteric site,
which brings about a conformational change in the
regulatory enzyme; affects the regulatory enzymes
active site, increasing (activator) or decreasing (inhibitor)
its activity

4. Zymogen Activation A zymogen is an inactive

enzyme precursor. Activation of a zymogen converts it
into an active enzyme which catalyzes reactions;

Calupitan, C.| Candido, S.| Canto, G.| Capistrano, D.| Base, J.

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Biochemistry1.4 |Enzyme Classification & Kinetics

5. Induction or Repression of Protein Synthesis

- Reaction velocity increases as an enzyme approaches

its optimum pH. Beyond that point, reaction velocity
decreases.
-Difference enzymes have different optimum levels of pH
-pH affects enzymatic activity through
ionization/deionization of the active sites.
3. Substrate Concentration

J. Factors Affecting Enzymatic Activity

1. Temperature

- The rate of enzymatic reactions increase with

increasing temperature only until it reaches the optimum
temperature
-Temperatures above the optimum level will decrease
the reaction velocity
-The heat energy in increased temperature causes more
collisions between enzyme and substrate which
increases reaction velocity.
-When optimum temperature is exceeded, enzymes may
be denatured, which decreases reaction velocity
2. pH

-Increasing substrate concentration improves reaction

velocity until Vmax is achieved.
-At Vmax, all the enzymes become saturated and the
reaction velocity will no longer increase.
-Increasing substrate concentration beyond Vmax
decreases reaction velocity due to the excessive
amounts of substrate competing for an active site. Active
sites become blocked resulting in a decrease in enzyme
saturation
4. Co-factors
- The presence of cofactors (like Cl-, I-, Br-, Zn+2, Mg+2,
etc.) increase the rate of an enzyme-catalyzed reaction.
K. Clinical Application of Enzymes
Cardiac Enzymes as Markers for Myocardial
Infarction (MI)
1. Troponin (Troponin T and Troponin I isoforms)
a. Regulatory proteins involved in myocardial
contractility; very specific and preferred markers for
detecting myocardial cell injury, as in myocardial
infarction; not present in the serum of healthy
individuals.
b. Rises 3-6 hours after injury; peaks in 12-16 hrs;
stays elevated in 5-14 days.
2. Creatine Kinase
a. Begins to rise 4-6 hours after MI; peak at 24 hrs;
returns to normal in 3-5 days.
b. Isoenzymes
b.1. CK-MM fraction = found in skeletal muscle

Calupitan, C.| Candido, S.| Canto, G.| Capistrano, D.| Base, J.

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Biochemistry1.4 |Enzyme Classification & Kinetics

b.2. CK-MB fraction = found in heart muscle

b.3. CK-BB = found in the brain
c. 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
a. Peak level about 36-40 hrs after MI and thus of
diagnostic value in patients admitted more than 48 hrs
after infarction.
b. Levels return to normal in 5-14 days.
c. 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.
4. Aspartate Aminotransferase, AST
a. Rise within 8 hrs after MI; peak at 24-36 hrs;
returns to normal preinjury level within 3-7 days.
b. Not diagnostic for MI since the enzyme is also
found in hepatocytes.
Enzyme Markers for Liver Disease Aminotransferases
a. Alanine Transaminase, ALT ( Serum GlutamatePyruvate Aminotransferase, SGPT)
- found predominantly in the hepatocytes, hence
diagnostic of liver pathology.
b. Aspartate Aminotransferase, AST (Serum
Glutamate-Oxaloacetate Aminotransferase, SGOT)
- also found in cardiac muscles, hence may be
helpful in the diagnosis of cardiac injury, as in
myocardial infarction.
c. When assaying for both ALT and AST, the ratio of
the level of these two enzymes can also be
diagnostic.
c1. In a liver damage not of viral etiology, ALT/AST
ratio is less than 1.
c2. In viral hepatitis, the ratio is greater than 1.

d. decrease enzyme concentration

2. An allosteric modification influences enzyme activity
by?
a. further decreasing activation energy
b. increases/decreases its activity
c. produces another active site on the enzyme
d. shuts down enzymatic activity

3. Which levels of protein organization is broken up

when optimum temperature is exceeded?
a. primary and secondary
b. Secondary and tertiary
c. tertiary and quaternary
d. primary and quaternary
4. Protein part of an enzyme that is catalytically
inactive. This describes:
a. Holoenzyme
b. Cofactor
c. Apoenzyme
d. Substrate
5. True or false: Enzymes can only catalyse one type of
chemical reaction.
ANSWERS: (1) a, (2) b, (3) b, (4) c, (5) true

V. References
Devlin, T. (2011). Textbook of Biochemistry with Clinical
Correlations, Wiley-Liss 7th Edition. New York: A John
Wiley & Sons, Inc., Publication.
Madarcos, F. (2014). Enzymes. Quezon City,Philippines.

IV. Guide Questions

1. How can the effects of competitive inhibition be
mitigated?
a. Increase substrate concentration
b. decrease substrate concentration
c. increase enzyme concentration

Calupitan, C.| Candido, S.| Canto, G.| Capistrano, D.| Base, J.

A.Y. 2014-2015

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Biochemistry1.4 |Enzyme Classification & Kinetics