Principles of clinical enzymology

Principles of Clinical Enzymology
Course: Clinical Laboratory Principle (SIMS-443)
ZA School of Medical Technology
1
Dr. Ali Raza
Senior Lecturer
SIMS-SIUT

Principles of Clinical Enzymology
 Introduction:
 Enzyme definition
 Composition:
 Protein part (Apoprotein)
 Non-protein(cofactors/coenzymes)
 Application
Enzyme Nomenclature
Basic Structure of Enzyme
 Homo-multimers
 Hetero-multimers
 Multiple Forms of Enzymes
 Origins of Enzyme Variants: Genetic and Non-genetic
 Example of Genetic and Non-genetic
 Iso-enzymes: Examples
 Specificity of Enzymes 2

Enzyme
“ A protein molecule that catalyzes chemical
reactions without itself being destroyed or
altered”
Catalyst:
A substance that increases the rate of a chemical
reaction, but is not consumed or changed by it.
An enzyme is a biocatalyst.
3

Holoenzyme:
• The functional compound
formed by the combination
of an
a) Apoenzyme
b) Coenzyme
4

Enzyme
a) Apoenzyme:
 The protein part of an enzyme without the cofactor
necessary for catalysis.
b) Coenzyme:
 A Diffusible, heat-stable substance of low molecular
that, when combined with inactive protein called an
Apoenzyme,
 helper molecules
5

Examples of Coenzyme:
 Coenzymes:
 Non-protein organic molecules
 Many (not all) are vitamins or are derived from vitamins
 Thiamine pyrophosphate (TPP)
 Flavin adenine dinucleotide (FAD), Biotin
Described as: Cosubstrates or Prosthetic groups.
• Cosubstrates: Coenzymes that bind tightly to a protein, yet will be released and
bind again at some point.(temporarily)
• Prosthetic groups: Enzyme partner molecules that bind tightly or covalently to
the enzyme
 Prosthetic groups permanently bond with a protein.
 An example of a prosthetic group is heme in hemoglobin, myoglobin,
and cytochrome.
6

Cofactor
 Inorganic species/ Non-protein compounds.
 Metal ions (Mg2+ , Mn2+ Ca2+
Important for nutrition:
Chromium,
Iodine,
Calcium
7

Enzyme Nomenclature
•The Enzyme Commission EC) of the International
Union of Biochemistry (IUB) developed a rational and
practical basis for identifying enzymes
•The number is prefixed by the letters EC, denoting
Enzyme Commission.
8

Nomenclature
All enzymes are assigned to one of six classes,
characterized by the type of reaction they catalyze:
(1) Oxidoreductases
(2) Transferases
(3) hydrolases
(4) Lyases
(5) Isomerases
(6) Ligases
9

Nomenclature
Capital letter abbreviations:
A common and convenient practice is to use abbreviations for
the names of certain enzymes
ALT = Alanine Aminotransferase
AST = Aspartate Aminotransferase
LD = Lactate dehydrogenase
CK = Creatine Kinase
G6PD =
14

Basic Structure of Enzyme
• All enzyme molecules possess the following level of
structural characteristics of proteins .
• Primary
• Secondary
• Tertiary
• Quaternary
15

Basic Structure of Enzyme
• Biological and catalytic activity requires two or more
folded polypeptide chains (subunits) to associate to
form a functional molecule (Quaternary structure).
• Homomultimers: Subunits may be copies of the same
polypeptide chain
E.g.: MM isoenzyme of Creatine kinase,
H4 isoenzyme of Lactate dehydrogenase
• Heteromultimers :
Represent distinct polypeptides.
16

Isoenzyme
One of a group of related enzymes catalyzing the same
reaction but having different molecular structures and
characterized by varying physical, biochemical, and
immunological properties.
18

Isoenzyme
lactate dehydrogenase is a tetramer made of two
different sub-units,
• H-form
• M-form
These combine in different combinations depending on
the tissue:
LDH1= HHHH
LDH2=HHHM
LDH3=HHMM
LDH4 =HMMM
LDH5=MMMM
19

Isoenzyme
• All the forms of a particular enzyme retain the
ability to catalyze its characteristic reaction.
• Have significant quantifiable differences in catalytic
activity.
• These enzyme variants may occur within a single
organ or even within a single type of cell.
21

Multiple Forms of Enzymes
Origins of Enzyme Variants could be
• Genetic
• Nongenetic
23

Multiple Forms of Enzymes
Genetic Origins of Enzyme Variants
• Due to the existence of more than one gene locus
coding for the structure of the enzyme protein are
called as True isoenzymes
• Many human enzymes (1/3) have more than one
structural gene locus.
24

Genetic Origins of Enzyme Variants may be
(a) Genes at the different loci have undergone modifications
during the course of evolution
(b) Isoenzymes are not necessarily closely linked on one
chromosome; they are often located on different
chromosomes
25

Genetic Origins of Enzyme Variants may be
(c) Oligomeric enzymes and consist of molecules made up of
subunits.
The association of different types of subunits in various
combinations gives rise to a range of active enzyme
molecules.
When the subunits are derived from different structural genes,
either multiple loci or multiple alleles, the hybrid molecules so
formed are called hybrid isoenzymes.
26

Genetic Origins of Enzyme Variants
 Enzymes of clinical importance that exist as
isoenzymes because of the presence of multiple gene
loci are
 Lactate dehydrogenase
 Creatine kinase
 alpha-amylase
 Alkaline phosphatase
27

Nongenetic Causes of Multiple Forms of Enzymes
• Posttranslational modification of enzyme molecules
give rise to multiple forms, known as isoforms
• Modification of the residues in the polypeptide
chains of enzyme molecules
• Changes affecting non-protein components of
enzyme molecules may also contribute to
molecular heterogeneity.
28

Modification of the residues in the polypeptide chains
of enzyme molecules includes
• Acylation
• Alteration of carbohydrates side chain
• Partial cleavage of chain
• De-amination
• Sulfhydryl Oxidation
• Phosphorylation
• Association with other proteins
29

Nongenetic modifications: Give rise to Isoforms
30

Example of Nongenetic Causes of Isoforms
• Removal of Amide groups accounts for Amylase and Carbonic
Anhydrase
• Adenosine deaminase, Acid phosphatase, Phosphoglucomutase,
contain sulfhydryl groups,
• susceptible to oxidation resulting in variant enzyme molecules
with altered molecular charge.
31

Many enzymes are glycoproteins, and variations in carbohydrate
side chains are a common cause of non homogeneity
of preparations of these enzymes.
N-acetyl neuraminaic acid (sialic acid)
32

• N-acetylneuraminic acid (sialic acid),
• are strongly ionized and consequently have a profound effect
on some properties of enzyme molecules.
• For example, removal of terminal sialic acid groups from human
liver and/or bone alkaline phosphatase with neuraminidase
greatly reduces the electrophoretic heterogeneity of the
enzyme.
33

Distribution of isoenzymes
• Not uniform throughout the body,
• Wide variations in the isoenzymes activity are found
at the organ, cellular, and subcellular levels.
• Tissue-specific differences are also found in the
distributions of some isoforms that are not due to
the existence of multiple gene loci.
34

Changes in lsoenzyme Distribution During
Development and Disease
• Multiple gene loci and their resultant isoenzymes
provide a means for the adaptation of metabolic
patterns to the changing needs of different organs
and tissue in the course of normal development or
in response to environmental change.
• Pathological conditions also are known to be
associated with alterations in the activities of
specific isoenzymes.
35

Examples of Changes in lsoenzyme
• The patterns of several sets of isoenzymes change
during normal development in tissue.
• During embryonic development of skeletal muscle,
LD and CK, progressively increase until the sixth
month of intrauterine life
• In early fetal development, three Aldolase
isoenzymes, A, B, and C, have been detected in
extracts of liver.
• At birth-as in the adult liver aldolase B is the
predominant isoenzyme. 36

Progressive muscular dystrophies appear to involve a
failure of the affected tissues to mature normally or to
maintain a normal state.
The distributions of isoenzvmes of aldolase., LD,. and
CK in the muscles of patients with progressive muscular
dystrophy have been found to be similar to those in the
earlier stages of development of fetal muscle.
The isoenzyme abnormalities in dystrophic muscle
have been interpreted as a failure to reach or maintain
a normal degree of differentiation.
37

Differences in Properties Between Multiple Forms of Enzymes
Genetic vs NonGenetic
The structural differences between the multiple forms of an
enzyme give rise to greater or lesser differences in
physicochemical properties
(1) Electrophoretic mobility,
(2) Resistance to inactivation
(3) Solubility, or in catalytic characteristics,(Ratio of reaction
with substrate analogs or response to inhibitors)
38

Differences in Properties Between Multiple Forms of Enzymes
Genetic vs NonGenetic
Genetic
Isoenzymes differ in catalytic properties
• molecular activity,
• K,, values for substrate(s),
• sensitivity to various inhibitors,
• relative rates of activity with substrate
analogs.
Immunological cross-reaction is not
uncommon
Differences in resistance to denaturation are
commonly found between true isoenzymes
NonGenetic
(posttranslational modifications)
• Have similar catalytic properties.
• Common antigenic determinants
39

Specificity of Enzymes
One of the properties of enzymes that makes them so important as diagnostic
and research tools is the specificity they exhibit relative to the reactions they
catalyze.
In general, there are four distinct types of specificity:
1. Absolute specificity - the enzyme will catalyze only one reaction.
2. Group specificity - the enzyme will act only on molecules that have specific
functional groups, such as amino, phosphate and methyl groups.
3. Linkage specificity - the enzyme will act on a particular type of chemical
bond regardless of the rest of the molecular structure.
4. Stereochemical specificity - the enzyme will act on a particular steric or
optical isomer
40

Enzyme Denaturation
• The catalytic activity of an enzyme molecule
depends on the integrity of its structure.
• Any disruption of the structure is accompanied
by a loss of activity, a process known
as denaturation.
41

Enzyme Denaturation
The partial or total alteration of the structure
of a protein, without change in covalent
structure, by the action of certain physical
procedures (heating, agitation) or chemical
agents.
42

Enzyme Denaturation
• Denaturation is either reversible or irreversible.
• Prolonged or severe denaturing conditions result in
an irreversible loss of activity.
• Denaturing conditions include
(1) Elevated Temperatures
(2) Extremes of pH
(3) Chemical addition
43

Enzyme Denaturation
(1) Elevated Temperatures:
a) At room temperature
b) Above 60 C
• Polymerase is an exception retain activity (90C)
• Storage: Low temperatures are used to preserve
enzyme activity
44

Enzyme Denaturation
2. Extremes of pH:
• Cause unfolding of enzyme molecular structures
• Should be avoided when preserving enzyme
samples.
45

Enzyme Denaturation
(3) Chemical Addition:
• Addition of chemicals disrupts
• hydrogen bonds and
• hydrophobic interactions
• Exposure of enzymes to strong solutions of these
reagents results in inactivation.
• Example: Urea
46

Enzymes as Catalysts
• Enzymes are protein catalysts of biological origin.
• Virtually, all chemical reactions that take place in
living matter are catalyzed by specific enzymes.
• Life itself is regarded as an integrated series of
enzymatic reactions and some diseases as a
derangement of the normal pattern of metabolism.
47

Enzyme Efficiency
• Biologically, a given number of enzyme molecules convert
an enormous number of substrate molecules to products
within a short time.
• Increased amounts of enzymes in the blood stream is
easily detected
• Amount of enzyme protein released from damaged cells
is small compared with the total level of non-enzymatic
proteins in blood.
• A particular enzyme is recognized by its characteristic
effect on a given chemical reaction
48

Specificity and the Active Center of Enzyme
Interaction between the enzyme and its substrate
involves the combination of one molecule of enzyme
with one substrate molecule (or two, bisubstrate
reactions).
Active Center :
The reaction involves the attachment of the substrate
molecule to a specialized region of the enzyme
molecule, its Active Center.
49

• Various groups are important in substrate binding are
brought together at the active center, and there the
processes of activation and transformation of the
substrate take place.
• The composition and spatial arrangement of the
active center also form the basis for the specificity of
an enzyme.
50

Active Site
The active site of an enzyme is/are
1. Relatively small (<5% of the total amino acids)compared
with the total volume of the enzyme molecule
2. 3-D structures are formed as a result of the overall
tertiary structure of the protein.
• This results from the amino acids and co-factors in
the active site of an enzyme being spatially structured
in an exact 3D relationship with respect to one
another and the structure of the substrate molecule.
51

The active site of an enzyme is/are
The attraction between the enzyme and its substrate molecules
is non-covalent binding.
Physical forces used in this type of binding include
(1) Hydrogen bonding
(2) electrostatic and hydrophobic interactions
(3) Van der Waals forces.
4. Occur in clefts and crevices in the protein, excludes bulk
solvent and reduces the catalytic activity of the enzyme.
5. The specificity of substrate binding is a function of the exact
special arrangement of atoms in the enzyme active site that
complements the structure of the substrate molecule.
52

Enzyme Kinetics
53
Catalysis of peptide bond hydrolysis by Chymotrypsin

Enzyme kinetics
Chemical kinetics, also known as reaction kinetics, is
the study of rates of chemical processes
54

Enzyme Kinetics
Basic Enzyme Reactions
Enzymes are catalysts and increase the speed of a chemical reaction without
themselves undergoing any permanent chemical change.
They are neither used up in the reaction nor do they appear as reaction products.
The basic enzymatic reaction can be represented as follows
where E = the enzyme catalyzing the reaction,
S = the substrate, the substance being changed,
P = the product of the reaction.
55

Energy Levels
The energy of activation:
• The quantity of energy is needed by chemical reactions to proceed.
• Magnitude of the activation energy which determines just how fast
the reaction will proceed.
• Enzymes lower the activation energy for the reaction they are
catalyzing.
56

The energy of activation
The enzyme is thought to reduce the "path" of the reaction. This shortened path
would require less energy for each molecule of substrate converted to product.
Given a total amount of available energy, more molecules of substrate would be
converted when the enzyme is present (the shortened "path") than when it is
absent. Hence, the reaction is said to go faster in a given period of time
57

The Enzyme Substrate Complex
A theory to explain the catalytic action of enzymes was proposed
by the Swedish chemist Savante Arrhenius in 1888.
He proposed that the substrate and enzyme formed some
intermediate substance which is known as the enzyme substrate
complex.
The reaction can be represented as:
58

The Enzyme Substrate Complex
At Yale University, Kurt G. Stern observed spectral shifts in catalase as the
reaction it catalyzed proceeded.
This experimental evidence indicates that the enzyme first unites in some
way with the substrate and then returns to its original form after the
reaction is concluded.
59

Enzyme kinetics
• The quantitative measurement of the (a) Rates of enzyme-
catalyzed reactions and (b) Factors that affect these rates.
• Rate of a chemical reactions:
Number of molecules of reactant(s) are converted into
product(s) in a specified time.
• Reaction rate is dependent on the
(a) Concentration of the chemicals
(b) Rate Constants of the reaction
60

• Enzymes act through the formation of an enzyme-substrate
(ES) complex, a molecule of substrate is bound to the active
center of the enzyme molecule.
• The binding process transforms the substrate molecule to its
activated state.
• ES complex breaks down to give the Products (P) and free
Enzyme (E):
• Activation energy takes place without the addition of external
energy so that the energy barrier to the reaction is lowered
Enzyme Kinetics
62

• Analytically, several enzymatic reactions may be linked
together to provide a means of measuring the activity of the
first enzyme or the concentration of the initial substrate in
the chain.
• When a secondary enzyme-catalyzed reaction, known as an
indicator reaction, is used to determine the activity of a
different enzyme, the primary reaction catalyzed by the
enzyme to be determined must be the rate-limiting step.
• Conditions are chosen to ensure that the “ rate of reaction
catalyzed by the indicator enzyme is directly proportional to
the rate of product formation in the first reaction.
Enzyme Kinetics
63

The International Unit (U)of Enzyme Activity
The EC of the IUB proposed
“ The quantity of enzyme that catalyzes the reaction of 1 μmol of
substrate per minute “
• Expressed in terms of U/L
• The catalytic activity of an enzyme is independent of the
volume, the unit used for enzymes is usually turnover per unit
time, expressed in katal (kat, mol s–1).
• The international unit U is still more commonly used
(μmol turnover min–1)
1 U = 10-6 mol/60s
64

Factors Governing the Enzyme Catalyzed Reactions
65

Factors Governing the Catalyzed Reactions
Factors that affect the rate of enzyme-catalyzed reactions
include
a) Enzyme concentration
b) Substrate concentration
c) pH
d) Temperature
e) Presence of Inhibitors
f) Presence of Activators
g) Coenzymes
h) Prosthetic groups
66

A- Enzyme Concentration
• Overall rate of the reaction is proportional to the [ES complex]
• Addition of more enzyme molecules to the reaction system
increases the conc. of the ES complex and the overall rate of
reaction.
• This increase accounts for the rate of reaction being proportional
to the concentration of enzyme present in the system
• Basis for the quantitative determination of enzymes by
measurement of reaction rates.
67

A- Enzyme Concentration
• As the amount of enzyme is
increased, the rate of reaction
increases.
• If there are more enzyme
molecules than are needed,
adding additional enzyme will
not increase the rate.
• So, Reaction rate increases as
enzyme conc. increases but
then it levels off.
68

B- Substrate Concentration
• Dependence of reaction rate on enzyme conc. under excess
substrate is present,
• Formation of an ES complex also accounts for the hyperbolic
relationship between reaction velocity and substrate
concentration
69

B- Substrate Concentration
• Plot of Substrate Concentration versus Reaction Velocity
Michaelis-Menten Plots
70

Substrate Concentration
• Single-Substrate Reactions
• Two -Substrate Reactions
• Consecutive Enzymatic Reactions
71

(a) Single-Substrate Reactions
First Order Reaction
Zero-Order Reaction
72

First Order Reaction
 At lower Substrate concentrations, the active sites on
most of the enzyme molecules are not filled.
 Higher concentrations cause more collisions
between the molecules. The rate of reaction increases
(First order reaction).
 Rate of the reaction is proportional and dependent
on the substrate concentration
73

Zero Order Reaction
• The maximum velocity of a reaction is reached when the
active sites are almost continuously filled.
• Increased substrate conc. after this point will not increase
the rate.
• At high substrate concentrations, the reaction rate is known
as zero-order reaction and is independent of substrate
concentration.
74

Maximal Velocity (Vmax)
• Reflects how fast the enzyme can catalyze the reaction
low Vmax enzymes
high Vmax enzymes
75

Michaelis Constant (Km)
• Substrate conc. that gives the enzyme one-half of its Vmax
low Km enzymes
high Km enzymes
https://www.chem.wisc.edu/deptfiles/genchem/netorial/modules/biomolecules/modules/enzymes/enzyme4.htm
76

Michaelis Constant (Km)
• Enzymes have varying tendencies (affinities) to bind their
substrates.
High Km
• A lot of substrate must be present to saturate the enzyme
• Enzyme has low affinity for the substrate.
Low Km
• A small amount of substrate is needed to saturate the enzyme,
• Enzyme has high affinity for substrate.
77

Km and its significance
Helps in determines
• the affinity of an enzyme for its substrate,
E.g: low the Km, higher is the affinity for substrate
• True substrate conc. for the enzyme.
 Zero-order kinetics are maintained if the substrate is present
in large excess 10 - 100 times value of Km.
 [S] = 10 x Km, [v is ~ 91% of the theoretical Vmax. ]
 The majority of enzymes Km are 10-5 to 10-3 mol/L
78

Km is Specific and constant for a given enzyme under
defined conditions of time , temperature and pH
79

Km, is reserved for the experimentally determined value
of [S] at which the reaction proceeds at one half of its
maximum velocity (v = Vmax/2).
80

The Michaelis-Menten
• It is a quantitative description of the relationship among
the rate of an enzyme-catalyzed reaction [v], the
concentration of substrate [S] and two constants, V max
and km
v = Reaction Rate
V max = Maximum Reaction Rate
S = Substrate Concentration
Km = The Michaelis-Menten Constant
81

Draws back of The Michaelis-Menten
• it is straightforward to set up an experiment to determine the
variation of v with [S]
• Hyperbolic curves: The exact value of Vmax is not easily
determined.
• Deviation from ideal behavior : Many enzymes at high substrate
conc. indeed may be inhibited by excess substrate
82

Line weaver- Burk plot
(Double Reciprocal )
83

Line weaver- Burk plot
(Double Reciprocal )
A Linear Form of the Michaelis-Menten Equation Is Used to
determine
• km
• V max
84

Lineweaver- Burk plot
• Plot of 1/vi as y as a function of 1/[S] as x therefore gives a
straight line
• whose y intercept is 1/ V max and whose slope is km/V max.
85

Lineweaver- Burk plot
With intercepts at l/Vmax on the ordinate (Y-axis) and -
1/K,, on the abscissa(X-axis).
86

Km
• It is now routine practice to determine kinetic constants,
such as Km and Vmax using a software package.
• When setting up methods of enzyme assay, it is necessary to
(1) Explore the relationship between reaction velocity and
substrate concentration over a wide range of concentrations
(2) Determine Km
(3) Detect any inhibition at high substrate Conc.
87

The optimal concentrations of substrate cannot be
used
(a) Substrate limited solubility
(b) substrate inhibits the activity of another enzyme
88

Two-Substrate Reactions
(Bisubstrate)
• Bisubstrate reactions are important in clinical enzymology
• The second substrate is a specific coenzyme,
 Reduced Nicotinamide-adenine dinucleotide (NADH)
 Reduced NAD Phosphate (NADPH)
Examples:
• Dehydrogenases
• Aminotransferases
89

(Bisubstrate)
90

(Bisubstrate)
91

Two-Substrate Reactions (Bisubstrate)
• The concentrations of both substrates affect the rates
of two-substrate reactions.
• Values of Km and Vmax, for each substrate are derived
from experiments
• Concentration of the first substrate is held at
saturating levels, whereas the concentration of the
second substrate is varied, and vice versa.
92

• The choice of substrate concentrations is limited by such
considerations as the
(1) solubility of the substrates,
(2) viscosity and high initial absorbance of conc. solutions,
(3) relative costs of the reagents.
93

The selection of appropriate substrate concentrations is
only one of the factors to be considered in formulating an
optimal assay system for the measurement of specific
enzyme activity.
94

(Bisubstrate)
• Critical choices must also be made with respect to other,
frequently interdependent factors that affect reaction
rate, such as
• Concentrations of Activators
• Nature and pH of the Buffer system.
95

C- pH
• Enzymatic Activity has been observed at pH values as
low as 1.5 (Pepsin)
as high as 10.5 (ALP)
• Many of the enzymes in blood plasma show
maximum activity in vitro in the pH range from 7 - 8.
• Optimal pH for a given forward reaction may be
different from the optimal pH found for the
corresponding reverse reaction.
96

pH
The pH-dependence curve is a result of a
(a) Ionization of the substrate
(b) The extent of dissociation of certain key amino acid
side chains in the protein molecule, both at the
active center and elsewhere in the molecule.
• Both pH and ionic environment will also have an
effect on the 3D conformation of the protein
• Enzyme activity to such an extent that enzymes may
be irreversibly denatured at extreme values of pH.
98

pH
• The effects of pH on enzyme reactions is control by
buffer solutions.
• The buffer system must be capable of counteracting
the effect of adding the specimen to the assay system
• E.g.: Serum itself is a powerful buffer
• Effects of acids or bases formed during the reaction
(e.g., formation of fatty acids by the action of lipase).
99

d - Temperature
• The rate of an enzymatic reaction is proportional to its reaction
temperature.
• Reaction rate increases with temperature to a max level,
then abruptly declines with further increase of temperature
102

Optimal temperature for enzyme activity
• Effects of the increased rate of the catalyzed reaction
and more rapid enzyme inactivation as the temperature
increases account for the existence of an apparent
optimal temperature for enzyme activity
103

D- Temperature
•A coefficient expressing the relation between a change in a
physical property and the change in temperature that causes it.
•Most animal enzymes rapidly become denatured at above 40C
• The temperature coefficient (Q10): The factor by which the
rate of a biologic process increases for a 10 °C increase in
temperature.
• For most enzymatic reactions, values of Q, (the
relative reaction rates at two temperatures differing
by 10 C) vary from 1.7 to 2.5.
104

Temperature
• The initial rate of reaction measured instantaneously
will increase with a rising temperature.
• A finite time is needed to allow the enzyme solution
to reach temperature equilibrium and to permit the
formation of a measurable amount of the product.
• During this period the enzyme is undergoing thermal
inactivation and denaturation a process that has a
very large temperature coefficient for most enzymes
and thus becomes virtually instantaneous at
temperatures of 60 "C to 70 "C.
105

Temperature
• An enzyme thermal inactivation is influenced by a
number of factors
(1) presence of substrate and its concentration,
(2) pH,
(3) Nature and ionic strength of the buffer.
106

Temperature
• Individual enzymes vary
• stability characteristics
• storage conditions
• Storage of serum samples at low temperatures is
necessary to minimize loss of enzyme activity while
awaiting analysis.
• Amylase is stable at room temperature (22-25C) for 24h
• Acid Phosphatase is exceedingly unstable, even when
refrigerated, unless kept at a pH below 6.0.
107

• Few enzymes are inactivated at refrigerator temperatures
• Example
liver-type isoenzyme of lactate dehydrogenase ( LD-5) =
less stable at lower temperatures.
• As a result, sera for LD determinations should be kept at
room temperature and not refrigerated.
108

E-Inhibitors and Activators
• Modifiers : their presence may reduces or increase the reaction rate
 Inhibitors
 Activators
• Small molecules
• Vary in specificity = Modifiers that exert similar effects on a wide
range of different enzymatic reactions at one extreme, to substances
that affect only a single reaction.
• Examples of nonspecific enzyme inhibitors
• Reagents, such as strong acids or multivalent anions and cations
that denature or precipitate proteins, destroy enzyme activity
109

Inhibitors
• Chemicals that reduce the rate of enzymic reactions
• Specific and work at low concentrations
• Block the enzyme but they do not usually destroy it
• Many drugs and poisons are inhibitors of enzymes in
the nervous system
• Inhibitors of the catalytic activities of enzymes provide
both pharmacologic agents and research tools for
study of the mechanism of enzyme action.
110

Inhibitors and Activators
• The activity of some enzymes depends on the presence of
particular chemical groups in the active center.
• E.g.: Reduced sulfhydryl(-SH) groups
• Inhibitors alter these groups of enzymes.
• E.g., oxidants of SH groups
111

Inhibitors and Activators
• Enzyme activation or inhibition are caused by
Interaction between the modifier and a non-enzymatic
component of the reaction system such as the substrate
• E.g.: Mg2+ combining with adenosine triphosphate (ATP)
to form Mg-ATP, required substrate for the CK reaction.
• In most cases, the modifier combines with the enzyme itself
in a manner analogous to the combination of enzyme and
substrate.
112

Enzyme Inhibition Classification
• Inhibitors can be classified based upon their site of
action on the enzyme,
• Chemically modify the enzyme
• Influence the kinetic parameters
113

Inhibition of Enzyme Activity
Inhibitors are classified as
• Reversible
• Irreversible
114

The effect of enzyme inhibition
• Irreversible inhibitors: Combine with the functional
groups of the amino acids in the active site
• Reversible inhibitors: These can be washed out of the
solution of enzyme by dialysis.
Applications of inhibitors
• Poisons snake bite, plant alkaloids and nerve gases
• Medicine antibiotics, sulphonamides, sedatives and
stimulants
• Negative feedback: end point or end product inhibition
115

Types of Enzyme Inhibition
• Competitive Enzyme Inhibition
• Non Competitive Enzyme Inhibition
• Uncompetitive Enzyme Inhibition
116

Enzyme Inhibition: Competitive vs Non-competitive vs
Uncompetitive
117

Reversible Inhibition
Reversible inhibition: Activity of the enzyme is
restored fully when the inhibitor physically is
removed from the system.
This type of inhibition is characterized by the
existence of an equilibrium between enzyme (E), and
inhibitor (I)
K, (the inhibitor constant), is a measure of the
affinity of the inhibitor for the enzyme,
118

Competitive inhibitor
• structural analog of the substrate: binds to the enzvme at the
substrate-binding site.
• Breakdown into products does not take place.
• When the process of inhibition is fully competitive, the
enzyme combines with either the substrate or the inhibitor,
but not with both simultaneously
119

Competitive Inhibitor
At low substrate concentrations, the binding of substrate
is reduced because some enzyme molecules are combined
with the inhibitor.
Conc. Of [ES] and the overall reaction velocity are reduced,
Km is increased.
At high substrate concentrations At high [S], all the
enzyme molecules combine to form ES so that
Vmax, is unaffected by the inhibitor.
120

A competitive inhibitor
• Structure similar to substrate(Structural Analog)
• Occupies active site
• Competes with substrate for active site
• Has effect reversed by increasing substrate Conc.
• Vmax remains same
• Km is increased
121

Competitive Inhibitor (C)
These characteristics of competitive inhibition are
demonstrated in the Line weaver-Burk plot
123

Competitive Inhibitor
• In two-substrate reactions, high concentrations of the
second substrate may compete with the binding of the
first substrate.
• Competitive inhibition contributes to the
• Reduction rate of an enzymatic reaction with time
• Nonlinearity of reaction progress curves.
124

Non-Competitive Inhibitor
• Structurally different from the substrate.
• Bind at a site on the enzyme other than the substrate-
binding site;
• No competition exists between
• Inhibitor and substrate
• enzyme-substrate-inhibitor (ESI) complex forms.
• Attachment of the inhibitor to the enzyme does not alter
the affinity of the enzyme for its substrate (K is unaltered),
•ESI complex does not break down to provide products.
125

Noncompetitive inhibitor
bind enzymes at sites distinct from the substrate-binding
site
• Generally bear little or no structural resemblance
to the substrate
• Binding of the inhibitor does not affect binding of
substrate
• Formation of both EI and EIS complexes is
therefore possible
• The enzyme-inhibitor complex can still bind
substrate, its efficiency at transforming substrate
to product, reflected by Vmax, is decreased
126

Non- competitive inhibitor
128

Noncompetitive inhibitor
Because the substrate does not compete with the
inhibitor for binding sites on the enzyme molecule,
an increase in the substrate concentration
does nut overcome the effect of a noncompetitive
inhibitor.
Thus Vmax is reduced in the presence
Km is not altered
129

Noncompetitive inhibitor (NC)
130

Noncompetitive inhibitor (NC)
• In the presence of a competitive
inhibitor,
Vmax can still be reached if sufficient
substrate is available, onehalf Vmax
requires a higher [S] than before and
thus Km is larger.
• With noncompetitive inhibition,
enzyme rate (velocity) is reduced
for all values of [S], including Vmax
and onehalf Vmax but Km remains
unchanged
131

Examples of Non-Competitive Inhibitor (NC)
• Cyanide inhibits cytochrome oxidase
• Fluoride inhibits Enolase and hence glycolysis
• Iodoacetate inhibits enzymes having SH groups in their active
sites
• BAL ( British Anti Lewisite, dimercaprol) is used as an antidote
for heavy metal poisoning
• Heavy metals act as enzyme poisons by reacting with the SH
groups
• BAL has several SH groups with which the heavy metal ions
bind and thereby their poisonous effects are reduced
132

Uncompetitive Inhibition (UC)
Uncompetitive inhibition: Due to combination of the
inhibitor with the ES complex and is more common in
two-substrate reactions, in which a ternary ESI
complex forms after the first substrate combines with
the enzyme.
parallel lines are obtained when plots of 1/v against
l/[S] with and without the inhibitor are compared
Both Km, and Vmax,, are decreased.
133

Inhibitor binds to enzymesubstrate complex
• Both Vmax and Km are decreased
• e.g ; Inhibition of placental alkaline phosphatase
(Regan isoenzyme) by Phenylalanine
134

135

Irreversible inhibition
• Render the enzyme molecule inactive by covalently and
permanently modifying a functional group required for
catalysis.
• Its effect is progressive with time, becoming complete if the
amount of inhibitor present exceeds the total amount of
enzyme.
• The rate of the reaction between enzyme and inhibitor is
expressed as the fraction of the enzyme activity that is
inhibited in a fixed time by a given concentration of
inhibitor.
• The velocity constant of the reaction of the inhibitor with
the enzyme is a measure of the effectiveness of the
inhibitor. 136

Irreversible inhibition
• Antienzymes: Important category of irreversible enzyme
inhibition
• E.g.: Trypsin inhibitors
• These are proteins that bind to trypsin irreversibly,
nullifying its proteolytic activity. E.g: Alpha 1-globulin
• Proteolysis inhibitors : Present in plasma prevent the
accumulation of excess thrombin / coagulation enzymes,
keeping the coagulation process under control.
137

Inhibition by Antibodies
• Enzyme-antibody complex has no effect on catalytic
activity
• Reaction of the enzyme and antibody reduces or stops
enzymatic activity.
• b/c antibody molecule (a)restricts access of the
substrate molecules to the active center by steric
hindrance
(b) completely masks the substrate-binding site
(c)induced conformational change in the enzyme 138

F- Enzyme Activation
• Activators increase the rates of enzyme-catalyzed reactions
by a variety of mechanisms of activation.
• For example: enzymes contain metal ions as an integral
part of their structures to stabilize tertiary and quaternary
protein structures.
• Removal of divalent metal ions by ethylene diamine tetra
acetic acid (EDTA) solution is accompanied by
conformational changes with inactivation of the enzyme.
• The enzyme often is reactivated by adding the ion to the
reaction mixture.
139

• Enzyme may be deficient in the ion so that addition of the
ion increases the reaction rate or indeed may be essential
for the reaction to take place.
• E.g.: Phosphate transfer enzymes (Creatine kinase) require
Mg2+ Ions.
• Activating Cations : Mn2+, Fez+, Ca2+, Zn2+, and K+.
• Activating Anions : Amylase functions at its maximal rate
in monovalent anions CI-, Br- or NO3-
140

• Some enzymes require the obligate presence of two
activating ions.
• K+ and Mg2+ are essential for Pyruvate Kinase,
• Mg2+ and Zn" are required for ALP activity.
141

Cofactors
Cofactors can be subdivided into two groups:
• Small organic molecules
• Metals
142

Coenzymes
• Small organic molecules
• Smaller than the enzyme proteins
• E.g: NAD, and NADP, are classified as coenzymes and are specific substrates in two-
substrate reactions.
• Their effect on the rate of reaction follows the Michaelis-Menten Pattern of
dependence on substrate concentration.
• Coenzymes such as NAD and NADP are bound only momentarily to the enzyme
during the course of reaction, as isthe case for substrates in general.
• Therefore no reaction takes place unless the appropriate coenzyme is present in the
solution.
143

Cofactors
• In contrast to these entirely soluble coenzymes, some
coenzymes are more or less permanently bound to the
enzyme molecules, where they form part of the active
center and undergo cycles of chemical change during
the reaction.
Prosthetic groups :
• Most common cofactor are also metal ions
• If tightly bound, the cofactors are called prosthetic groups
• The prosthetic group may be organic (such as a
vitamin, sugar, or lipid) or inorganic (such as a metal
ion), but is not composed of amino acids.
144

Prosthetic Groups
• The active holoenzyme results from the combination of the inactive apoenzyme with the
prosthetic group.
• An example of a prosthetic group is pyridoxal phosphate (P-5'-P), a component of AST and ALT.
• The P-5'4' prosthetic group undergoes a cycle of conversion of the pyridoxal moiety to
pyridoxamine and back again during the transfer of an amino group from an amino acid to an oxo-
acid.
Prosthetic groups, such as activators with a structural role, do not usually have to be added to elicit
full catalytic activity of the enzyme unless previous treatment has caused the prosthetic group to be
lost from some enzyme molecules.
both normal and pathological serum samples contain appreciable amounts of apo-
aminotransferases,which is converted to the active holoenzymes by a suitable period of incubation
with P-5'-P.
145

pH
• Buffers have their maximum buffering capacity
close to their pK, (-log ionization constant K,) values,
• A buffer system should be chosen with a pKa, value
within 1 pH unit of the desired pH of the assay.
146

148
Reference:
Tietz Fundamentals of Clinical Chemistry,
Sixth Edition., Principles of Clinical Enzymology
Chapter 9, pg. 140-154

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