ENZYMOLOGY

GENERAL PROPERTIES AND

DEFINITION OF TERMS

Enzymes
A protein that acts as a catalyst.
Biological polymers that catalyzes the chemical
reaction.
Enzymes are neither consumed nor permanently
altered as a consequences of their participation in
a reaction
Extremely selective catalysts.

 As a protein, each enzyme contains a specific

amino acid sequence (primary structure), with the
resultant polypeptide chains twisting (second
structure), which then folds (tertiary structure) and
results in structural cavities.
If an enzyme contains more than one polypeptide
unit, the quaternary structure refers to the spatial
relationships between the subunits.

Apoenzyme
The protein part of an enzyme without the cofactor
necessary for catalysis. The cofactor can be a metal ion, an
organic molecule (coenzyme), or a combination of both.
Coenzyme

The dissociable, low-relative-molecular-mass active group

of an enzyme which transfers chemical groups, hydrogen,
or electrons. A coenzyme binds with its associated protein
(apoenzyme) to form the active enzyme (holoenzyme).

Holoenzyme
An active enzyme consisting of the apoenzyme
and coenzyme.
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.

Catalyst
A catalyst is a substance that increases the rate of
a reaction without modifying the overall standard
Gibbs energy change in the reaction; the process is
called catalysis, and a reaction in which a catalyst is
involved is known as a catalyzed reaction
Catalytic activity
The property of a catalyst which is measured by the
catalyzed rate of conversion of a specified chemical
reaction, produced in a specified assay system.

 Catalytic activity concentration

The property of an enzyme obtained by dividing
the catalytic activity by the volume of the original
system from which the sample comes.
Induction
In enzymology, induction is a biological process
which results in an increased biosynthesis of an
enzyme thereby increasing its apparent activity. It
results from the presence of an inducer.

 Inhibition
An inhibitor is a substance that diminishes the rate of a
chemical reaction; the process is called inhibition.
Activation energy
An operationally defined quantity expressing the
dependence of a rate coefficient on temperature. In
enzymology it is usually considered to be the energy
required for a molecule to form an activated complex
which is in the transition of making or breaking a chemical
bond. In an enzyme-catalyzed reaction, this corresponds
to the formation of the activated enzyme-substrate
complex.

 Product
The substance produced by the enzymecatalyzed conversion of a substrate.
Substrate
A substrate is a reactant (other than the catalyst
itself) in a catalyzed reaction.

CLASSIFICATION AND
NOMENCLATURE

Classification
Oxidoreductases- catalyze oxidationreduction reactions
e.g., lactate dehydrogenase
Transferases- catalyze the transfer of C, N,
or P containing groups
e.g., creatinine kinase
Hydrolases- catalyze the cleavage of bonds
by addition of water
e.g., amylase

 Lyases- catalyze the cleavage C-C, C-S, and

certain C-N bond
e.g.,pyruvate decarboxylase

Isomerases- catalyze the racemization of

optical or geometric isomers
e.g., phosphohexose isomerase

Ligases- catalyzed the formation of bond

between carbon and O, S,N
e.g., pyruvate carboxylase

Nomenclature
Recommended name- usually short and convenient for
everyday use.
a) enzymes have names ending in suffix
-ase
b) describe the action performed by the
enzyme
c) represent trivial names

 Systematic name
-more complete than the recommended
name
- name assigned by the IUBMB for
enzyme
-all enzymes use the suffix -ase

 Enzyme has classification number made up of four digits

First digit- class name
Second digit- subclass
Third digit- sub-subclass
Fourth digit- specific number of enzyme for subsubclass

 Example:
Recommended name: Creatinine
kinase
ATP + creatinine= ADP +
phosphocreatinine
Systematic name: ATP:creatinine
phosphotransferase
Classification number is EC 2.7.3.2.

ENZYME KINETICS

Catalytic Mechanisms of Enzyme

A chemical reaction may occur if the
kinetic energy is higher for the reactants
than the products.
Activation energy
-Certain energy reactants need before they will
react

 Enzyme catalyst lower the energy barrier,

allowing the reactants to react faster
to form a product.
The ES complex- physical binding of a
substrate to the active site of enzyme

 The transition state for the ES complex

has a lower energy of activation than
the transition state of substrate alone,
so that the reaction proceeds after the
complex is formed.

 Enzymes are substrate specific.

absolute specificity- the enzyme combines with only
one substrate and catalyzed only one reaction.
Group specific- combine with all substrates
containing a particular chemical groups.

 Stereoisometric specificity- refers to enzyme that are

predominantly combine with only one optical isomer of a
certain compound.
An enzyme may bind with more than
one molecule of a substrate.
Binding of one substrate molecule may facilitate binding of
additional substrate molecule.

FACTORS THAT INFLUENCE

ENZYMATIC REACTIONS

ENZYME CONCENTRATION
The rate of reaction is generally proportional to the
amount of enzyme present.

SUBSTRATE CONCENTRATION
First Order Kinetics rate of the reaction is
proportional and dependent on the substrate
concentration.
Zero Order Kinetics it is independent of substrate
concentration

EFFECT OF pH
The rate of enzyme-catalyzed reactions typically
shows marked dependence on pH
Many of enzymes in blood plasma show maximum
activity in vitro in the pH range from 7 to 8
The optimal pH for a given forward reaction may be
different from the optimal pH for the corresponding
reverse reaction

TEMPERATURE
The rate of an enzymatic reaction is proportional to
its reaction temperature
However, an increase in the rate of the catalyzed
reaction is not the only effect of increasing
temperature on an enzymatic reaction
Enzymes denature at 60-70 degrees celsius

INHIBITORS and ACTIVATORS

Inhibitors reduces the rate of reaction
Activators increases the rate of reaction

COENZYMES and PROSTHETIC GROUPS

Coenzymes are more complex molecules than
activators
Coenzymes such as NAD and NADP are bound only
momentarily to the enzyme during the course of the
reaction, as is the case for substrates in general
Therefore, no reaction takes place unless the
appropriate coenzyme is present in solution

 In contrast to 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
When bound, the coenzyme is called prosthetic
group

MEASUREMENT OF
ENZYME ACTIVITY

 Enzymes are measured in terms of their activity rather

that their concentration per se

REACTION RATE
MEASUREMENT
Determination of reaction rate involves the kinetic
measurement of the amount of changed produced
within a defined time interval
Fixed time and Continuous-monitoring methods are
used to measure reaction rates

 Fixed time method

The amount of change produced by the enzyme
is measured after the reaction is stopped at the
end of a fixed time interval
Continuous-monitoring method
The progress of the reaction is monitored
continuously

 The progress of an enzyme reaction is monitored by

measuring in time the decreasing concentration of
substrate or the increasing concentration of the
products
At the moment when enzyme and substrate are
mixed, the rate of reaction is zero
The rate typically rises rapidly to a maximum value,
which remains constant for a period of time

 During the period of constant reaction rate, the rate

depends only on enzyme concentration and is
completely independent of substrate concentration
The reaction is said to follow zero-order kinetics
because its rate is proportional to the zero power of
the substrate concentration
However, as more substrate consumed, the reaction
rate declines and enters a phase of first-order
dependence on substrate concentration

SUBSTRATE MEASUREMENT
Starts with a high substrate concentration
The amount of substrate transformed into products
during an enzyme-catalyzed reaction can be
measured with any appropriate analytical method
such as spectrophometry, fluorometry, or
chemiluminescence

PRODUCT MEASUREMENT
This starts with zero initial product level
It is more accurate

COENZYME MEASUREMENT
Measures increase or decrease in coenzyme

ENZYME MASS CONCENTRATION

MEASUREMENT
Many immunoassays have been developed for
human enzymes and isoenzymes that measure
protein mass instead of catalytic activity
To develop such assays, purified enzyme protein has
to be prepared to act as a calibrator, be labeled,
and be used to raise the enzyme specific antibody.

 These methods identify all molecules with the

antigenic determinants necessary for recognition by
the antibody, so that inactive enzyme molecules
that are immunologically unaltered are measured
along with active molecules
This has been found to be significant in
determination of some digestive enzymes

TYPES OF ENZYME
ASSAYS

End-point analysis
Measurement of product or substrate is done at the
end of the reaction

Multi-point assay
Measures the change in the concentration of the
indicator substance at several intervals during the
course of the assay

Kinetic assay
Involves continuous measurement of change in
concentration as a function of time

Use of coupled reactions

Enzymatic activity is measured by coupling the
activity with colorimetric reaction
The colored product is measured
spectrophotometrically

Pitfalls in Conventional Enzyme

Assays
Hemolysis
Anticoagulants
Lactescence or Milky serum

Storage of Samples
Most enzymes are stable at 6 ^C for at least 24
hours and at room temperature for lesser periods
For prolonged storage, use 20^C or lower
Some enzymes are not preserved at -20^C and
must be kept at
-70^C (CK)
Few enzymes are inactivated at refrigerator
temperature

Quality Control Program for

Enzyme Assay
To ensure accuracy and reliability of enzyme tests,
the ff should be observed
1. Strict adherence to zero-order kinetics
2. Proportionality studies with increments of
samples
3. Use pooled frozen serum or stable reference
materials as controls
4. Replicate measurements to evaluate precision
of assays

ENZYMES AS
REAGENTS

 Enzymes are used as analytical reagents for

measurement of several metabolites and
substrates and in immunoassays to detect
and quantify immunological reactions

Measurement of Metabolites
The use of enzymes as analytical reagents to
measure metabolites frequently offers the
advantage of great specificity for the substance
being determined
The high specificity removes the need for
preliminary separation or purification stages, so the
analysis is carried out directly on complex mixture
such as serum

 Uricase (urate oxidase), urease and glucose

oxidase are examples of highly specific
enzymes used in clinically important assays
for measurement of uric acid, urea and
glucose specifically in biological fluids

Mechanisms for
Abnormal Serum
Enzyme Levels

Increased Serum levels

1.
2.
3.
4.
5.
6.
7.

Necrosis
Acute hepatitis
Acute pancreatitis
Increased membrane permeability (w/o necrosis)
Increased tissue source of enzyme
Increased enzyme synthesis
Impaired excretion of enzyme

Decreased Serum levels

Genetic
1. Hypophosphatasia
2. Wilsons disease
3. Acholinesterasemia
Acquired
1. Hepatitis
2. Starvation
3. Enzyme inhibition
4. Lack of cofactors

ENZYMES OF CLINICAL
SIGNIFICANCE

Creatine kinase
Enzyme classification: Transferases
E.C. code no: 2.7.3.2
Catalyzes the reaction:
Creatine + ATP creatine-phosphate + ADP

Creatine kinase
Tissue distribution:
Skeletal muscle
Heart muscle
Brain
Bladder
Placenta
lungs

Isoenzymes of Creatine Kinase

1. CK-BB (Brain Type): in BRAIN INJURY
2. CK-MB (Hybrid Type): in AMI
3. CK-MM (Skeletal Muscle Type): in MUSCLE INJURY

Factors that could interfere with

test results:
Hemolysis
Light
Anticoagulants
If the pxn is bed-ridden or physically trained

Creatine Kinase
Methods used:
Absorbance of NADPH (Oliver-Rosalki-Hess)
Decrease in absorbance (Tanzer & Gilvarg)

Lactate Dehydrogenase
Found in all tissues
Abundant in:
Myocardium
Kidney
Liver
skeletal muscles
RBCs

Lactate Dehydrogenase
Methods used:
Reaction with DNPH (Wroblewski-Cabaud)
Conversion of pyruvate to lactate (WroblewskiLaDue)
Conversion of lactate to pyruvate (Wacker)

Aspartate Aminotransferase
SGOT (Serum Glutamic Oxaloacetate Transferase)
Enzyme classification: Transferases
E.C. no.: 2.6.1.1
Reaction catalyzed:
aspartate + a-ketoglutarate oxaloacetate +
glutamate

Aspartate Aminotransferase
Tissue Distributions:
Cardiac muscle
Skeletal muscle
Liver mitochondria
Kidney
Pancreas
Erythrocytes

Aspartate Aminotransferase
Methods of AST Determination:
Reaction with dinitrophenylhydrazone (Frankel-Reitman)
Coupling with Diazonium salt (Babson Method)
Coupled Enzyme Reaction (Karmen or Walker)

Alanine Aminotransferase
SGPT (Serum Glutamic Pyruvic Transferase)
Enzyme classification: Transferases
E.C. no.: 2.6.1.2
Reaction catalyzed:
alanine + a-ketoglutarate pyruvate + glutamate

Alanine Aminotransferase
Tissue Distributions:
Liver
Kidney
Myocardium
Skeletal muscle
Pancreas

Spleen
Lung
Erythrocyte
Cytosol

Alanine Aminotransferase
Methods of ALT Determination:
Reaction with dinitrophenylhydrazone (Frankel-Reitman)
Coupling with Diazonium salt (Babson Method)
Coupled Enzyme Reaction (Karmen or Walker)

Acid Phosphatase
Enzyme classification: Hydrolases
E.C. no.: 3.1.3.2
Reaction catalyzed:
phosphomonoester + H2O alcohol + phosphate ion

Acid Phosphatase
Tissue distributions:
Prostate
Red cell
Platelet
Spleen
Liver
BM
kidney

Acid Phosphatase
Methods used:
Roy Method (most specific)
Bodansky
Gutman & Gutman King Armstrong
Hudson
Bessey-Lowry-Brock
Babson & Reed
Rietz- Guilbault

Gamma-glutamyltransferase
Enzyme involved in the transfer of the y-glutamyl
residue from y-glutamyl peptides to amino acid, H2O
and other small peptides.
Reaction sequence:
AST

Glutathione + amino acid

cysteinylglycine

Glutamyl peptide + L-

Clinical significance = Hepatic Disorder

Methods used = Szasz method
Enzyme Classification= Transferases

Amylase
Enzyme classification: Hydrolases
An enzyme that catalyzes the breakdown of starch
and glycogen.
Clinical significance: Pancreatitis

Lipase
An enzyme that hydrolyzes the ester linkages of fats
to produce alcohols and fatty acids.
Clinical significance: Acute pancreatitis

G6PD
Enzyme Classification: Oxidoreductases
An enzyme that function to maintain NADPH in
reduced form.
Clinical Significance: Drug induced hemolytic
anemia