ENZYMES

Winston S. Abena, MD, FPASMAP, COSH

Saint Paul University Philippines
School of Medicine

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 Outline

• Definition of enzyme terms and nomenclature

• Description of general properties of enzymes
• Binding energy and transition states
• Catalytic mechanisms and functional groups

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 Enzymes
• biologic polymers that catalyze multiple
dynamic processes
• play a central role in health and disease
• normal rate of turnover can be a basis for
clinical enzymology
• pharmacology & toxicology (competitive
inhibition)
• diagnosis of genetic diseases (recombinant
DNA technology) – PCR & Restriction
Fragment Length Polymorphism (RFLP)
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 Enzymes
• all reactions in the body are mediated by
enzymes (protein catalysts); the rate of
reactions without themselves being changed in
the process
• they selectively channel reactants
(intermediates), because the product of a
reaction becomes the substrate for the next
step…

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 Enzyme Nomenclature
• each enzyme is assigned two names
a) recommended name – convenient for everyday
use
- names have the suffix “-ase” attached to the substrate
of the reaction
e.g. glucosidase, urease, sucrase
- description of the action performed
e.g. lactate dehydrogenase, adenylate cyclase
- some retain their original trivial names
e.g. trypsin, pepsin
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 Enzyme Nomenclature

b) Systematic name – more complete; used when

the enzyme must be identified without
ambiguity
- this was developed by the International Union of
Biochemistry and Molecular Biology (IUBMB),
enzymes are divided into 6 major classes each with
numerous subgroups

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 Enzyme Nomenclature
I. Oxidoreductases – catalyze oxidation-reduction reactions e.g.
Lactate dehydrogenase
II. Transferases – catalyzes transfer of C, N, or P-containing groups
e.g. Serine hydroxy methyl transferase (serine  glycine)
III. Hydrolases – catalyze cleavage of bonds by addition of water
e.g. Urease (urea  CO2 + NH3)
IV. Lyases – catalyze cleavage of C-C, C-O, C-S, & certain C-N
bonds e.g. Pyruvate decarboxylase (pyruvate  acetaldehyde)
V. Isomerases – catalyzes racemization of optical & geometric
isomers e.g. Methylmalonyl coA, Mutase (Methylmalonyl coA 
Succinyl coA)
VI. Ligases – catalyze formation of bonds between C & O, S, N
coupled to hydrolysis of high energy PO4s
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12/25/23 8
 Enzyme Nomenclature
• active site - a region of an enzyme comprised of
different amino acids where catalysis occurs
(determined by the tertiary and quaternary structure
of each enzyme)
• substrate - the molecule being utilized and/or
modified by a particular enzyme at its active site
• co-factor - organic or inorganic molecules that are
required by some enzymes for activity. These
include Mg2+, Fe2+, Zn2+ and larger molecules
termed coenzymes like nicotinamide adenine
dinucleotide
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(NAD +
), coenzyme A, and many 9
vitamins.
 Common Coenzymes
Coenzyme Reaction mediated
Biotin Carboxylation
Cobalamin (B12) Alkylation transfers
Coenzyme A Acyl transfers
Flavin Oxidation-Reduction
Lipoic acid Acyl transfers
Nicotinamide Oxidation-Reduction
Pyridoxal Phosphate Amino group transfers
Tetrahydrofolate One-carbon group transfers
Thiamine
12/25/23 pyrophosphate Aldehyde transfer 10
 Enzyme Nomenclature
• prosthetic group - a metal or other co-enzyme
covalently bound to an enzyme
• holoenzyme - a complete, catalytically active
enzyme including all co-factors
• apoenzyme - the protein portion of a holoenzyme
minus the co-factors
• isozyme - (or iso-enzyme) an enzyme that performs
the same or similar function of another enzyme.
This generally arises due to similar but different
genes encoding these enzymes and frequently is
tissue-type
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specific or dependent on the growth 11or
developmental status of an organism.
 Enzyme Catalysis Overview

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 Properties of Enzymes
1. Active sites
2. Catalytic efficiency
– most enzyme-catalyzed reactions are highly efficient
– Increased reaction rates sometimes 10⁶ to 1012
increase
• Enzymes do not change ΔG just the
reaction rates
- typically each enzyme molecule is capable of
transforming 100 – 1,000 substrate molecules into
product each second
- the number of the molecules of substrate converted
to product per enzyme molecule per sec. = turnover
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 Properties of Enzymes
3. Specificity
- enzymes are highly specific interacting with one or a few
specific substrates & catalyzing only one type of chemical
reaction

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 Substrate specificity

The non-covalent bonds and forces are maximized to

bind substrates with considerable specificity
•Van der Waals forces
•electrostatic bonds (ionic interactions)
•Hydrogen bonding
•Hydrophobic interaction

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 Enzymes are Stereospecific
O
 
CH 3CH 2 OH  NAD  CH 3CH  NADH  H

Yeast
Alcohol dehydrogenase

Specific residues help

maintain stereospecificity

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 Geometric specificity
Selective about identities of chemical groups
but
Enzymes are generally not molecule specific
There is a small range of related compounds that will
undergo binding or catalysis.

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 Similar shaped molecules can be highly toxic

O
H
C
NH2

N
+
N CH3
N

Tobacco Nicotine

Because of this closeness in name

Nicotinic acid was renamed to niacin by the bread
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 Properties of Enzymes
4. Cofactors
5. Regulation
- enzyme activity can be regulated (activated or
inhibited); so that the rate of product formation
responds to the needs of the cell
6. Location within the cell
- localized in specific organelles;
a. serves to isolate the reaction substrate or product
from other competing reactions,
b. to provide a favorable environment for the
reaction, and
c. to organize the thousands of enzymes present in
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the cell 19
 ENZYMATIC REACTION
PRINCIPLES
• A catalyst is unaltered during the course of a reaction and
functions in both the forward and reverse directions. In a
chemical reaction, a catalyst increases the rate at which the
reaction reaches equilibrium, though it does not change the
equilibrium ratio. For a reaction to proceed from starting
material to product, the chemical transformations of bond-
making and bond-breaking require a minimal threshold
amount of energy, termed activation energy. Generally, a
catalyst serves to lower the activation energy of a particular
reaction.

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 ENZYMATIC REACTION
PRINCIPLES
• The energy maxima at which the reaction has the
potential for going in either direction is termed the
transition state. In enzyme catalyzed reactions, the same
chemical principles of activation energy and the free
energy changes (Go) associated with catalysts can be
applied. An overall negative Go indicates a favorable
reaction equilibrium for product formation. The net effect
of the enzyme is to lower the activation energy required
for product formation.

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 Chemical Reaction

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 Enzymatic Reaction Energetics

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 Reaction Rates
• The rate of the reaction is determined by several factors
including the concentration of substrate, temperature and pH.
For most standard physiological enzymatic reactions, pH and
temperature are in a defined environment (pH 6.9-7.4, 37 oC).
Therefore, the concentration of substrate is the critical
determinant. This enzymatic rate relationship has been
described mathematically by combining the equilibrium
constant (the ratio of substrate and product concentrations),
the free energy change and first or second-order rate theory.
The net result for enzymatic reactions is that the lower the
activation energy, the faster the reaction rate, and vice versa.
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 Binding Energy
• The graph of activation energy and free energy changes for an
enzymatic reaction also indicates the role binding energy
plays in the overall process. Due to the high specificity most
enzymes have for a particular substrate, the binding of the
substrate to the enzyme through weak, non-covalent
interactions is energetically favorable and is termed
binding energy. The same forces important in stabilizing
protein conformation (hydrogen bonding and hydrophobic,
ionic and van der Waals interactions) are also involved in the
stable binding of a substrate to an enzyme.

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 LOCK-AND-
KEY

INDUCED FIT

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 Binding Energy and Transition
State
• The cumulative binding energies from the non-covalent
interactions are optimized in the transition state and is the
major source of free energy used by enzymes to lower
activation energies of reactions. A single weak interaction has
been estimated to yield 4-30 kJ/mol energy, thus multiple
interactions (which generally would occur during binding and
catalysis) can yield up to 60-80 kJ/mol free energy - this
accounts for the large decreases in activation energies and
increases in rate of product formation observed in enzymatic-
catalyzed reactions.

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 Effect of Temperature
A reaction rate will generally
increase with increasing
Temperature due to increased
kinetic energy in the system until
a maximal velocity is reached.
Above this maximum, the kinetic
energy of the system exceeds the
energy barrier for breaking weak
H-bonds and hydrophobic
interactions, thus leading to
unfolding and denaturation of the
enzyme and a decrease in reaction
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rate.
 Effect of pH
Variations in pH can affect a
particular enzyme in many ways,
especially if ionizable amino acid
side chains are involved in binding
of the substrate and/or catalysis.
Extremes of pH can also lead to
denaturation of an enzyme if the
ionization state of amino acid(s)
critical to correct folding are
altered. The effects of pH and
temperature will vary for different
enzymes and must be determined
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experimentally. 30
 Kinetics of Enzymes
Enzymes follow zero order kinetics when substrate
concentrations are high. Zero order means there is no increase
in the rate of the reaction when more substrate is added.
Given the following breakdown of sucrose to glucose and
fructose
Sucrose + H20 Glucose + Fructose
H
H H
H OH O
OH
HO H OH
H O H
HO OH H
HO H H
HO
H OH

H OH

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 k1
E  S  ES  E  P
k2

k -1

E = Enzyme S = Substrate P = Product

ES = Enzyme-Substrate complex
k1 rate constant for the forward reaction
k-1 = rate constant for the breakdown of the ES to
substrate
k2 = rate constant for the formation of the
products
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 Assumption of equilibrium
k-1>>k2 the formation of product is so much
slower than the formation of the ES complex.
That we can assume:

k 1 E S
Ks  
k1 ES
Ks is the dissociation constant for the ES complex.

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 Assumption of steady state
Transient phase where in the course of a reaction the
concentration of ES does not change

d ES
0
dt

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 Michaelis-Menten Equation

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 Michaelis-Menten Curve

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 Initial Velocity (vo) and [S]
• The concentration of substrate [S] present will greatly
influence the rate of product formation, termed the
velocity (v) of a reaction. Studying the effects of [S] on
the velocity of a reaction is complicated by the
reversibility of enzyme reactions, e.g. conversion of
product back to substrate. To overcome this problem, the
use of initial velocity (vo) measurements are used. At the
start of a reaction, [S] is in large excess of [P], thus the
initial velocity of the reaction will be dependent on
substrate concentration

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 Initial Velocity (vo) and [S]
(cont)
• When initial velocity is plotted against [S],
a hyperbolic curve results, where Vmax
represents the maximum reaction velocity.
At this point in the reaction, if [S] >> E, all
available enzyme is "saturated" with bound
substrate, meaning only the ES complex is
present.

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 Substrate Saturation of an Enzyme

A. Low [S] B. 50% [S] or Km C. High, saturating [S]

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 Steady State Assumption
• The M-M equation was derived in part by making several
assumptions. An important one was: the concentration of
substrate must be much greater than the enzyme
concentration. In the situation where [S] >> [E] and at initial
velocity rates, it is assumed that the changes in the
concentration of the intermediate ES complex are very small
over time (vo). This condition is termed a steady-state rate,
and is referred to as steady-state kinetics. Therefore, it
follows that the rate of ES formation will be equal to the
rate ES breakdown.

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The Km is the substrate concentration where vo equals
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one-half Vmax 41
 Conclusions about Michaelis-Menten
Kinetics
1. Characteristics of Km
a. Small Km: a numerically small (low) Km reflects a high affinity of the
enzyme for substrate; because a low conc. of substrate is needed to half-
saturate the enzyme
b. Large Km: a numerically large (high) Km reflects a low affinity of enzyme
for substrate; because a high conc. of substrate is needed to half-saturate the
enzyme
2. Relationship of velocity to enzyme conc.:
The rate of the reaction is directly proportional to the enzyme conc. at all
substrate conc.
3. Order of Reaction:
First order – S < Km, velocity ≈ substrate conc.
Zero order – S > Km, velocity = Vmax
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 Two Substrate Reactions
• Many enzyme reactions involve two or more substrates.
Though the Michaelis-Menten equation was derived from a
single substrate to product reaction, it still can be used
successfully for more complex reactions.

Random

Ordered

Ping-pong
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 Two Substrate Reactions
• In random order reactions, the two substrates do
not bind to the enzyme in any given order; it does
not matter which binds first or second.
• In ordered reactions, the substrates bind in a
defined sequence, S1 first and S2 second.
• These two reactions share a common feature termed
a ternary complex, formed between E, ES1, ES2
and ES1S2. In this situation, no product is formed
before both substrates bind to form ES 1S2.
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 Two Substrate Reactions
• Another possibility is that no ternary
complex is formed and the first substrate S 1
is converted to product P1 before S2 binds.
These types of reactions are termed ping-
pong or double displacement reactions.

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 Lineweaver-Burk (double
reciprocal plot)
• If the reciprocal (1/X) of the Michaelis-Menten
equation is done, after algebraic simplification the
following equation results:

• This relation is written in the format of the equation

for a straight line, y = mx + b, where y = 1/v o, m
(slope) = Km/Vmax, x = 1/[S] and the y-intercept, b =
1/Vmax. When this relation is plotted,the result is a
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straight line graph
 Lineweaver-Burk (double
reciprocal plot)

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 Uses of double reciprocal plot
• The x intercept value is equal to -1/K m. The
biggest advantage to using the double
reciprocal plot is a more accurate
determination of Vmax, and hence Km. It is
also useful in characterizing the effects of
enzyme inhibitors and distinguishing
between different enzyme mechanisms.

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 Enzyme Inhibitor Types
• Inhibitors of enzymes are generally molecules
which resemble or mimic a particular enzymes
substrate(s). Therefore, it is not surprising that
many therapeutic drugs are some type of enzyme
inhibitor. The modes and types of inhibitors have
been classified by their kinetic activities and sites of
actions. These include Reversible Competitive
Inhibitors, Reversible Non-Competitive Inhibitors,
and Irreversible Inhibitors

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 Uses of Ki
• Ki values are used to characterize and compare
the effectiveness of inhibitors relative to Km. This
parameter is especially useful and important in
evaluating the potential therapeutic value of
inhibitors (drugs) of a given enzyme reaction. For
example, Ki values are used for comparison of the
different types of HIV protease inhibitors. In
general, the lower the Ki value, the tighter the
binding, and hence the more effective an
inhibitor is.
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 Competitive Inhibition

Vmax - No change
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K m INCREASES - indicates a direct interaction
51
of the inhibitor in the active site
 Reversible Competitive Inhibition
• Competitive inhibitors compete with the substrate
for binding at the active site (as E + I). In the double
reciprocal plot for a competitive inhibitor acting at
the substrate site for the following reasons, notice
with increasing concentration of inhibitor, the Vmax
does not change; however, the Km of the
substrate is increased. This also reflects the
reversible nature of the inhibitor; there is always
some concentration of substrate which can displace
the12/25/23
inhibitor. 52
 Non-Competitive Inhibition

Vmax DECREASES - inhibitor affects rate of reaction

by binding to site other than substrate active-site
Km - No change
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 Reversible Non-Competitive Inhibition
• Non-competitive inhibitors combine with both the enzyme
(E + I) and the enzyme-substrate (EI + S) complex. The
inhibitor binds to a site other that the substrate site, and is
thus independent of the presence or absence of substrate.
This action results in a conformational change in the
protein that affects a catalytic step and hence decreases or
eliminates enzyme activity (formation of P). Notice in the
reciprocal plot, a non-competitive inhibitor does not affect
the binding of the substrate (Km), but it does result in a
decrease in Vmax. This can be explained by the fact that
since inhibitor bound to an enzyme inactivates it, the more
EI formed will lower [ES] and thus lower the overall rate
of 12/25/23
the reaction Vmax. 54
 Irreversible Inhibitors
• Irreversible inhibitors generally result in the destruction or
modification of an essential amino acid required for enzyme
activity. Frequently, this is due to some type of covalent
link between enzyme and inhibitor. These types of
inhibitors range from fairly simple, broadly reacting
chemical modifying reagents (like iodo-acetamide that
reacts with cysteines) to complex inhibitors that interact
specifically and irreversibly with active site amino acids
(termed suicide inhibitors). These inhibitors are designed to
mimic the natural substrate in recognition and binding to an
enzyme active site. Upon binding and some catalytic
modification, a highly reactive inhibitor product is formed
that binds irreversibly and inactivates the enzyme.
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Irreversible Inhibitor: Allopurinol

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Irreversible Inhibitor: Penicillin (Ex)

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 Diisopropyl Phosphofluoridate:
Irreversible Acetylcholinesterase
Inhibitor (Example)

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 Clinical Use of Enzymes
• Enzyme Activity in Body Fluids Reflects Organ
Status:
• Cells die and release intracellular contents;
increased serum activity of an enzyme can be
correlated with quantity or severity of damaged
tissues (ex. creatine kinase levels following heart
attack)
• Increased enzyme synthesis can be induced and
release in serum correlates with degree of
stimulation (ex. alkaline phosphatase activity as a
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liver status marker)
 Enzymes in Clinical Diagnosis
Elevated Serum Disease Diagnosed
Enzymes
Creatine Kinase Myocardial Infarction
Amylase & Lipase Acute pancreatitis
Alanine aminotransferase Liver damage
Acid phosphatase Cancer of the prostate
Alkaline phosphatase Bone & Liver disease
Lactate dehydrogenase Myocardial infarction,
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Hemolytic anemias 60
 Clinical Use of Enzymes
• Enzyme Activity Reflects the Presence of
Inhibitors or Activators
• Activity of serum enzymes decreases in presence
of an inhibitor (ex. some insecticides inhibit serum
cholinesterases)
• Determine co-factor deficiencies (like an essential
vitamin) by enzyme activity (ex. add back vitamin
to assay, if activity increases, suggests deficiency
in that vitamin)
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 Enzymes good as Therapeutic Drugs
Drugs Enzymes Disease indicated
Inhibited
Acyclovir DNA polymerase Herpes simplex

Zidovudine (AZT) Reverse AIDS

transcriptase
Rifampicin RNA polymerase Tuberculosis

Chloramphenicol Peptidyl Bacterial

transferase infections

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 Therapeutic Agents: Thrombolytic
Drugs
Streptokinase Tissue Plasminogen
Activator (t-PA)
Less selective for fibrin clot More selective
Produces plasminemia (-)
Reduces mortality Reduces mortality
Causes allergic reaction (-)
Causes hypotension (-)
Less expensive More expensive
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 Clinical Use of Enzymes
• Enzyme activity can be altered genetically
• A mutation in an enzyme can alter its substrate affinity, co-factor
binding stability etc. which can be used as a diagnostic in
comparison with normal enzyme
• Loss of enzyme presence due to genetic mutation as detected by
increased enzyme substrate and/or lack of product leading to a
dysfunction
• NOTE: PCR techniques that identify specific messenger RNA or
DNA sequences are replacing many traditional enzymatic based
markers of genetic disease

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 Enzymes in Genetic Abnormalities
Inborn Error of Deficient Enzymes
Metabolism
Phenylketonuria Phenylalanine hydroxylase
Albinism Tyrosinase
Maple syrup urine disease α-keto acid dehydrogenase
Gaucher’s disease Glucocerebrosidase
Niemann-Pick’s disease Sphingomyelinase
Tay-Sach’s disease Hexosaminidase A

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 Enzymes in Clinical Diagnosis
Elevated Serum Disease Diagnosed
Enzymes
Creatine Kinase Myocardial Infarction
Amylase & Lipase Acute pancreatitis
Alanine aminotransferase Liver damage
Acid phosphatase Cancer of the prostate
Alkaline phosphatase Bone & Liver disease
Lactate dehydrogenase Myocardial infarction,
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Hemolytic anemias 66
 Isoenzymes (Isozymes)
Isozymes
• enzymes that catalyze the same reaction but
differ in their physical properties because of
genetically determined differences in AA
sequence
• different organs contain characteristic
proportions of different isoenzymes
• the pattern of isoenzymes found in the plasma
may serve as a means of identifying the site of
tissue damage
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 Isoenzymes (Isozymes)
Isozymes
• they may contain different numbers of charged
AAs and may be separated from each other by
electrophoresis
• e.g. Creatine kinase – 3 isoenzymes w/ 2
polypeptides
CK1 – BB, CK2 – MB, CK3 – MM
*each isoenzyme show a characteristic
electrophoretic mobility

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 Diagnosis of Myocardial Infarction
• Myocardial muscle – is the only
tissue that contains more than 5% of
the total CK activity as the CK2
(MB) isoenzyme
• appearance of CK2 in plasma is
virtually specific for infarction of
the myocardium
• it appears approximately 4 – 8 hrs
following onset of chest pain &
reaches a peak of approx. 24 hrs infarction
• LD may also be elevated peaking
only 3 – 6 days after the onset of
symptoms
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 LD1 (HHHH)
LD2 (HHHM)
LD3 (HHMM)
LD4 (HMMM)
LD5 (MMMM)

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12/25/23 71
 Enzymes Used in recombinant DNA
research
1. Restriction endonucleases
- recognizes short segments of DNA
- cleaves double-stranded DNA into smaller fragments
2. TAG polymerase – amplifies DNA through
polymerase chain reaction (PCR)
3. Reverse transcriptase – synthesizes DNA from
RNA template
4. DNA ligase – catalyze bond formation between
DNA fragments
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Enzymes Used in recombinant DNA
research

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