CLINICAL MEDTECH LEC

CHEMISTRY II TERM 02

CLINICAL ENZYMOLOGY

INTRODUCTION AND MECHANISM

Enzymes are biologic proteins that
catalyze biochemical reactions that is not
consumed nor changed in composition
These are catalysts that can be used in the
monitoring and diagnosis of disease, and
their remarkable properties make them
sensitive indicators of pathologic change.
Found in all body tissue (intracellular) and
is increased in serum after cell injury COMPONENTS OF AN ENZYME
Physiologically, enzymes are Active Site
predominantly at low concentrations and
▪ A cavity of a enzyme where
low activity, and increase in serum
substrates bind and undergo a
activities of these enzymes are used to
chemical reaction
infer the location and nature of pathologic
changes in tissues of the body. ▪ Maybe or maybe not specific for a
particular substrate
An understanding of the factors that affect
the rate of release of enzymes from their ▪ Relatively smaller than the total size
cell of origin and at which they are cleared of the enzyme.
from the circulation is necessary to
interpret correctly changes in activity that Allosteric Site
occur with disease. ▪ A cavity other than the active site
that binds regulatory (effective)
FUNCTIONS OF ENZYMES
molecules
▪ Hydration of carbon dioxide (respiration)
▪ Inhibitors binds to the allosteric to
▪ Nerve Induction control the activity of catalytic
reaction.
▪ Muscle contraction
ACTIVE AND ALLOSTERIC SITE
▪ Nutrient Degradation (digestion)
▪ Growth and Reproduction
▪ Energy Storage and Use

GENERAL PROPERTIES OF ENZYME

Enzyme catalyze many specific
physiologic reactions.
These reactions are facilitated by the Terms Associated with Enzymes
enzyme structure and several other Substrates
factors.
▪ Substances acted upon enzymes
As a protein, each enzyme contains a
specific amino acid sequence (primary ▪ Specific for each of their particular
structure) with resultant polypeptide enzyme
chains twisting (secondary structure), ▪ Lipase: Triglycerides; Amylase: Starch
which then folds (tertiary structure) and
results in structural cavities. Cofactors

If an enzymes contains more than one ▪ Non protein substances added in the
polypeptide unit, the quarternary enzyme-substrate complex to manifest
structures refers to the spatial the enzyme activity
relationship between the subunits

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▪ Coenzyme: An organic cofactor that Isoenzymes and Isoforms contribute to

hastens enzymatic reaction, [Link] in properties and functions of
momentarily bound (e.g. NAD and enzymes.
NADP)
Holoenzyme
▪ Prosthetic Groups or Activators:
▪ An active substance formed by
Usually a metal ion ( e.g. calcium, zinc,
combination of a coenzyme and an
chloride, magnesium and potassium)
apoenzyme, usually a combination of
▪ When bound tightly to the enzyme, the apoenzyme and prosthetic group.
coenzyme is called prosthetic group.
Proenzyme or Zymogens
Substrates
▪ An inactive enzyme precursor
▪ Substances acted upon enzymes
▪ E.g. coagulation factors and digestive
▪ Specific for each of their particular enzymes (prothrombin, pepsinogen)
enzyme
▪ Lipase: Triglycerides; Amylase: Starch

Cofactors
▪ Non protein substances added in the
enzyme-substrate complex to manifest
the enzyme activity
▪ Coenzyme: An organic cofactor that ENZYME CLASSIFICATION AND NOMENCLATURE
hastens enzymatic reaction, With International Union of Biochemistry
momentarily bound (e.g. NAD and (IUB) system, a systematic and trivial
NADP) name is provided for each enzyme. The
▪ Prosthetic Groups or Activators: systematic name describes the nature of
Usually a metal ion ( e.g. calcium, zinc, the reaction catalyzed and is associated
chloride, magnesium and potassium) with unique numeric code.

▪ When bound tightly to the enzyme, the Consists of 4 numbers separated by

coenzyme is called prosthetic group. periods (e.g. [Link])

Isoenzyme The first number defines the class to which

the enzyme belongs. All enzymes are
▪ Enzymes with similar enzymatic assigned to one of six classes,
activity but differ in their physical, characterized by the type of reaction they
biochemical and immunologic catalyze:
characteristics
▪ 1 – Oxidoreductase
▪ Helps in in understanding of organ-
specific patterns of metabolism. ▪ 2 – Transferases

▪ Examples of enzymes that exists as ▪ 3 – Hydrolases

isoenzymes because of presence of ▪ 4 – Lyases
multiple-gene loci are LD, CK, ALT, etc.
▪ 5 – Isomerases
Apoenzyme
▪ 6 – Ligases
▪ The protein portion of the enzymes
subject to denaturation in which EC NUMBER
enzyme losses its activity
▪ Minimal denaturation maybe reversed
by removal of denaturing reagent.
▪ Affected by extreme pH, temperature
and chemical addition.
Isoforms
Results when an enzyme is subject to
posttranslational modifications.

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EC SYSTEMATIC NAME TRIVIAL NAME ABBR.

NUMBER

[Link] L-lactate: NAD Lactate dehydrogenase LD

oxidoreductase

[Link] L-glutamate: NAD(P) Glutamate dehydrogenase GLD

oxidoreductase

[Link] (γ-glutamyl)- γ-glutamyltransferase GGT

peptide: amino acid
γ-
glutamyltransferase

[Link] L-aspartate: 2- Aspartate AST

oxoglutarate aminotransferase
aminotransferase (transaminase)

[Link] L-alanine: 2- Alanine aminotransferase ALT

oxoglutarate (transaminase)
aminotransferase

[Link] ATP: creatine N- Creatine kinase CK

phosphotransferase

[Link] Triacylglycerol Lipase LPS

acylhydrolase

[Link]. Orthophosphoric- Alkaline phosphatase ALP

monoester
phosphohydrolase
(alkaline)

[Link] 5’-ribonucleotide 5’-nucleotidase NTP

phosphohydrolase

[Link] D-fructose-1,6- Aldolase ALD

biphosphate D-
glyceraldehyde-3-
phosphate-lyase

ENZYME CLASSIFICATION AND NOMENCLATURE ▪ Catalyze the transfer of a group

(phosphate, methyl, etc.) other than
1 - Oxidoreductases hydrogen between two substrates
▪ Catalyze redox reaction between two ▪ A-X + B 🡪 A + B-X
substrates
▪ E.g. transferase (ALT, AST, GGT) and
▪ A+B🡪A+B kinase (CK)
▪ E. g. Dehydrogenase (LD, G6PD), CO,
MDH, ICD
2 – Transferases

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▪ A🡪B
▪ E.g. Glucose phosphate isomerase and
ribose phosphate isomerase
6 - Ligases
▪ Catalyze the joining of two substrate
molecules, coupled with breaking of
pyrophosphate bond in ATP
▪ AB + C 🡪 A-C + B

▪ Glutathione Synthetase (GSH-S)

3 - Hydrolases
▪ Catalyze hydrolysis or splitting of a bod
by the addition of water (hydrolytic
reactions)
▪ A-B + H₂O 🡪 OH – B-H

▪ E.g. Esterases (ACP, ALP, CHS, LPS)

▪ Peptidases (Trypsin, Pepsin, LAP)
▪ Glycosidase (AMS, Galactosidases)
4 - Lyases
▪ Catalyze breakdown of groups from
substrates without hydrolysis; the
product remain double bonds
▪ ATP 🡪 cAMP + PP¡
▪ Glutamate decarboxylase, Pyruvate
decarboxylase, Tryptophan
decarboxylase and aldolase

ENZYME KINETICS
Enzymes act through the formation of an
enzyme-substrate complex, in which a
molecule of substrate is bound to the
active center of the enzyme molecule.
The binding process transforms the
substrate molecule to its activated process
transforms the substrate molecule to its
activated state.

Energy needed for transformation is

provided by the free energy binding of
substrate to enzyme, therefore activation
takes place without addition of external
energy, so that the energy is lowered and
5 - Isomerases the process is accelerated.
▪ Catalyze the intramolecular Catalytic Mechanisms of Enzymes
arrangement of the substrate
compound Factors that Influence Enzymatic
Reactions

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Measurement of Enzyme Activity Substrate Concentration

Calculation of Enzyme Activity ▪ Michaelis and Menten assumed that
equilibrium is attained rapidly among
CATALYTIC MECHANISMS OF ENZYMES enzyme, substrate and enzyme-
Enzymes catalyze physiologic reactions by substrate complex with the effect of
lowering the activation energy level that product formation (ES🡪P) on the
the reactants must reach concentration of ES being negligible.
▪ Provided an excess of free substrate
molecules is present, addition of more
enzyme molecules to the reaction
system, increases the concentration of
ES complex, thus the overall rate of
reaction.
Substrate Concentration

First Order Kinetics

▪ Reaction rate proportional to the
substrate concentration
▪ The reaction rate is directly
Relationship between enzyme, substrate proportional to substrate
and product are in theory reversible. concentration.
E + S 🡪 ES 🡪 E + P Zero Order Kinetics
▪ Only a fixed number of substrate (in
excess) is converted to product per
seconds.
▪ The reaction rate depends only on
enzyme concentration.

Absolute specificity
▪ Enzyme combines with only one
substrate and catalyzes only one
corresponding reaction

Group specificity
▪ Combine with all substrates containing
a particular chemical group
pH
Bond specificity
▪ Controlled by buffers
▪ Specific for chemical bonds
▪ Most shows maximum activity in
Stereoisomeric specificity vitro in the pH range of 7.0 to 8.0

▪ Enzymes that combine with only one ▪ However, activity has been observed
optical isomer at pH as low as 1.5 (pepsin) and as
high as 10.5 (ALP)
Factors that Influence Enzymatic
Reactions Temperature

Enzyme Concentration ▪ Enzymes are active at 25C, 30C, or 37C

▪ The higher the enzyme concentration, ▪ 37C is the optimum temperature for
the faster is the reaction because more enzymatic activity.
enzyme is present to bind with the
▪ Increasing temperature usually
substrate.
increases the rate of a chemical

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reaction by increasing the movement coenzyme, or undergoing oxidation or

of molecules. reduction.

▪ The rate of denaturation increases as Common Activators:

the temperature increases, and is
Metallic: Calcium, Iron, Magnesium,
usually significant at 40C to 50C
Manganese, Zinc and Potassium
▪ 60-65C may result inactivation of
Non-metallic – Bromide, Chloride
enzyme
Cofactors
▪ Temperature Coefficient (Q10) means
for every 10C increase in temperature ▪ These are nonprotein entities that must
there will be a two-fold increase in bind to particular enzymes before a
enzyme activity. reaction occurs.
Cofactors b. Coenzymes- is an organic compound
(second substrates)
▪ Coenzymes and prosthetic groups
increases the rates of enzyme- - when bound tightly to the enzyme,
catalyzed reactions by promoting conenzyme are called prosthetic groups.
formation of the most active state of
the enzyme or other reactants, such - increasing coenzyme concentration will
as the substrate. increase the velocity of an enzymatic reaction
in a manner synonymous with increasing
Inhibitors substrate concentration.
▪ Inhibition are classified as reversible - it is essential to achieve absolute
or irreversible. enzymatic activity.
▪ Reversible inhibitor implies that the - example” calcium, zinc, chloride,
activity of the enzyme is fully magnesium and potassium
restored when the inhibitor is
removed from the system in which c. Metalloenzymes- are inorganic ion
the enzyme acts by some physical attached to a molecule
process, such as dialysis, gel - example- catalase and cytochrome
filtration or chromatography. oxidase.
▪ Irreversible inhibitor combines Inhibitors
covalently with the enzyme so that
physical methods are ineffective in ▪ Enzymatic reactions may not progress
separating the two. Examples are normally if a particular substance, an
organophosphates are extremely inhibitor, interferes with the reaction.
potent inhibitor against esterases
a. Competitive Inhibitor
such as acetylcholinesterase.
- Physically bind to the active site of an
Cofactors
enzyme and compete with the
▪ These are nonprotein entities that substrate for the active site.
must bind to particular enzymes
- Both the substrate and inhibitor
before a reaction occurs.
compete for the same active site of the
▪ Activators- are inorganic ions which enzyme with a substrate
alters the spatial configuration of the concentration significantly higher
enzyme for proper substrate than the concentration of the inhibitor,
binding. The activator may be the inhibition is reversible.
essential for the reaction or may
- The effect of the inhibitor can be
only enhance the reaction rate in
counteracted by adding excess
proportion with concentration to
substrate to bind the enzyme. Dilution
the point at which excess activator
of serum results to reduction in the
begins to inhibit reaction. Activator
concentration of this inhibitor, thus
function by alternating spatial
increasing the rate of reaction.
configuration of the enzyme for
proper substrate binding, linking - It has the ability to alter the apparent
substrate to the enzyme or Michaelis-Menten constant.

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Inhibitors - Room temperature= ideal storage of

LDH (LD4 and LD5)
▪ Enzymatic reactions may not progress
normally if a particular substance, an Hemolysis – mostly increases enzyme
inhibitor, interferes with the reaction. concentration
b. Non-Competitive Inhibitor Lactescense or milky specimens- decrease
enzyme concentration.
▪ It does not compete with the substrate but
look for areas other than the active site. MEASUREMENT OF CATALYTIC ACTIVITY
▪ The substrate and inhibitor (commonly Measurement of enzyme reaction is can be
metallic ion) may bind an enzyme measured by the following changes in
simultaneously because the inhibitor factors and reactions.
binds the enzyme independently from the
substrate, increasing substrate Increase in product concentration – the
concentration does not reverse the amount is directly proportional to the
inhibition. decrease in substrate.

Inhibitors Decrease in substrate concentration

▪ Enzymatic reactions may not progress Decrease in coenzyme concentration

normally if a particular substance, an (NADH) – amount of generated coenzymes is
inhibitor, interferes with the reaction. usually applied in coupled reactions and
optical changes.
b. Uncompetitive Inhibitor
Increase in altered enzyme concentration
▪ The inhibitor binds to the enzyme-
substrate (ES) complex. Dependent on enzyme concentration

▪ Increasing the substrate concentration Always performed in zero-order kinetics

results in more ES complexes to which the • the moment the enzyme and
inhibitor binds and thereby increases the substrate is mixed, the reaction is
inhibition. zero. The rate typically rises rapidly
▪ Increasing the substrate concentration to maximum value which remains
results to increase inhibitors. constant for a period of time.
During this period, the rate
depends only on the enzyme
concentration and is completely
independent of substrate
concentration.
• Measures the final product formed
during a fixed period of incubation
in which the rate remains constant.
Measured during the linear phase of
reaction
General Methods of Measuring Enzymatic
Reaction

• Storage Fixed Time (Two-Point) Assay

- Low temperature (refrigeration/freezing) ▪ The reagents are combined; the reaction

render enzymes reversibly inactive. proceeds, the reaction is stopped and the
amount of reaction is measured
- Repeated freezing and thawing tends
to denature proteins and should be Continuous Monitoring or Kinetic
avoided. Assays

- -20C = preservation for longer period • Multiple measurements at specific time

of time (enzymes) intervals or continuous measurement as
absorbance changes
- 2C to 8C -= ideal storage temperature
for substrate and coenzymes ▪ Deviation from zero kinetics can be
observed

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UNITS FOR EXPRESSING ENZYME ACTIVITY

IU
▪ The amount of enzyme that will catalyze the
reaction of 1 umol of substrate per minute
(umol/min)
Kat (SI)
▪ The amount of enzyme that will catalyze the
reaction of 1 mol of substrate per second
(mol/s)

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