ENZYMES

Definition
Largely ‘protein’ in nature produced
by living cells
Biologic catalysts
Measured in terms of their activity and
NOT in terms of their absolute values.
 Large molecules and normally
confined within cells unless increased
membrane permeability allows them
to enter the blood.
Frequently appear in the serum after
cellular injury, degradation of cells or
from storage areas.
 Clinically, abnormal large amounts
of enzymes in serum: evidence of
organ damage.
 Each catalyzes a single reaction or
a limited number of chemical
reactions, and it is specific for a
substrate that it converts to a defined
product.
 General Properties of
Enzymes
Each enzyme contains:
1.ACTIVE SITE
= water-free cavity, where the substrate
interacts with particular charged amino
acid residues (3-dimensional protein
structure)
= specific portion of enzyme that binds
on the substrate
2. ALLOSTERIC SITE
= a cavity other than the active site which
may bind with regulatory molecules.
Enzyme Theories
1. Emil Fisher’s/ Lock& Key
Theory=
 Enzyme have a “rigid” active
site
 The shape of the key(substrate)
must fit into the lock(enzyme).
Enzyme Theories
2. Kochland’s/ Induced Fit Theory
=
 Enzyme have a ‘flexible” active
site
 Based on the substrate binding
to the active site of the enzyme.
Enzyme Kinetics
Enzymes work by lowering
activation energy
◦ Activation energy
◦ Transition state
Enzyme do their works, action of
the enzyme
Enzyme Kinetics
Chemical Reaction:
◦ Downhill movement of energy
◦ High energy of the reactants going
to the lower energy of product
Types of
Specificity

 Absolute specificity
 An enzyme combines with only one substrate
and catalyzes only one reaction.
 Group specificity
 Enzymes combine with all the substrates in a
chemical group.
 Bond specificity
 Enzymes reacting with specific chemical
bonds.
 Stereoisomers
 Isoenzyme/Isoforms
 Michaelis Menten
Hypothesis
Ifthe reaction reached the
maximum velocity, no reaction
will happen

V= velocity
Vmax= maximum
velocity
Km= Michaelis
constant
S= Substrate
Enzymatic Reactions
1. Zero-order reaction
 reaction rate depends only on
enzyme concentration.
 Happens when “ S>E”
 Occurs during Vmax

2. First-order reaction
 reaction rate is directly proportional
to a substrate concentration.
 Happens when “E>S”
 Factors affecting Enzyme
Reactions
1. Enzyme Concentration
2. Substrate Concentration
3. Cofactors
4. Inhibitors
5. Isoenzymes
6. Temperature
7. Hydrogen Ion Concentration or pH
8. Storage
9. Hemolysis- increases enzyme
concentration
10. Lactescence or Milky specimen-
decreases enzyme concentration
1. Enzyme Concentration
The HIGHER the enzyme
concentration, the FASTER is the
reaction, because MORE enzyme
is present to bind with the
substrate.
 2. Substrate
Concentration
With the amount of enzyme
exceeding the amount of substrate,
the reaction rate steadily
INCREASES as more substrate is
ADDED.
However, when the substrate
concentration reaches a MAXIMAL
VALUE, higher concentration of
substrate no longer result in
increased rate of reaction.
 3. Cofactors
 Non-protein entities that must bind to particular
enzymes before a reaction occurs.
a. Co-enzymes (ex. NADP, NADH)
◦ Organic compound (2nd substrates)
◦ Increasing coenzyme conc. increase the
velocity of an enzymatic reaction
b. Activators (ex. Calcium, Zinc, Chloride)
◦ Inorganic ions
◦ alter spatial configuration of the enzyme for
proper substrate binding
c. Metalloenzymes (Ex. Catalase)
◦ Inorganic ion attached to a molecule.
 4. Inhibitors
Enzymatic reactions may not
progress if an inhibitor interferes
with the reaction.
Inhibition as to reversibility
a. Reversible
b. Irreversible
Inhibition as to activity
a. Competitive
b. Non-competitive
c. Uncompetitive
 1. Competitive Inhibitor
Physically bind to the active site
of an enzyme.
Both the substrate and the
inhibitor compete for the same
active site of the enzyme.
Reversible: adding more
substrate
 2. Non-competitive
Inhibitor
Binds an enzyme at a place other
than the active site.
Do not compete with the
substrate but look for areas other
than the active site.
Irreversible: not influence by
adding kore substrate
3. Uncompetitive Inhibitor
The inhibitor binds to the Enzyme
Substrate (ES) complex, so that
increasing substrate conc.
Results in more ES complexes to
which the inhibitor binds and
thereby increases the inhibition.
 5. Isoenzymes
These are enzymes having the
same catalytic reactions but
slightly different molecular
structures
These different forms occur due
to their differences in amino acid
sequences of enzymes.
 6. Temperature
 Increasing temperature usually increases
the rate of chemical reaction by increasing
the movement of molecules.
 ACTIVE- 25C, 30C, or 37C
 OPTIMUM TEMPERATURE- 37C
 RATE OF DENATURATION – 40-50C
 INACTIVE- 60-65C, ref and freezing temp.
 Temperature Coefficient (Q10)/ Vant Hoff’s
Rule
◦ For every 10C increase in temperature, there
will be a two-fold increase in enzyme activity
◦ With limit until 50-60oC
7. Hydrogen Ion Concentration
or pH
Extreme pH level may denature an
enzyme or influence its ionic state
resulting in structural change or in
the charge of amino acid residue in
the active site.
Most physiologic reactions occur in
the
pH 7-8.
 8. Storage
-20C =preservation for longer
period of time.
2-8C= IDEAL TEMP. FR
SUBSTRATE & COENZYMES
Room temp. =IDEAL FOR
STORAGE OF LDH (LD4&LD5)
 Classification of Enzymes
CLASS FUNCTION EXAMPLES
1. Oxidoreductase Catalyzes the removal or CO, LDH, MDH, ICD,
addn of electrons.(redox rxn)
s G6PD
2. Transferases Catalyzes the transfer fo a CK, AST, ALT
chemical group other than
Hydrogen from one
substrate to another.

3. Hydrolases Catalyzes the hydrolysis or ESTERASES

splitting of a bond by the ACP, ALP,
addn of water. (hydrolytic
rxn)

4. Lyases Catalyzes removal of groups
from substrates w/o
hydrolysis. The product
contains DOUBLE BONDS
PEPTIDASES
TRYPSIN, PEPSIN, LAP
GLYCOSIDASES
AMS, Galactosidase
Glutamate decarboxylase,
Pyruvate decarboxylase,
Tryptophan decarboxylase,
Aldolase

5. Isomerases Catalyzes the intermolecular Glucose phosphate

arrangement of the substrate isomerase, Ribose phosphate
cmpd isomerase
 Enzyme Nomenclature
The ENZYME COMMISION (EC) adapted a
classification system in 1961 and revised
the standards in 1972 and 1978 in order
to standardize enzyme nomenclature.
FIRST DIGIT= “Classifications”
SECOND & THIRD DIGITS= “Subclass”
FINAL & FOURTH NUMBER/S= “Serial no.”
Examples:
◦ Acid Phosphatase E.C 3.1.3.2
◦ Creatine Kinase E.C. 2.7.3.2
Enzyme Activity
Enzymes are measured in terms
of:
1.Change in the SUBSTRATE
CONCENTRATION
2. Change in PRODUCT
CONCENTRATION
3. Change in COENZYME
CONCENTRATION
 Two general methods used to
measure the extent of enzymatic
reactions
1. Fixed-time aka End point Method
 Rxn takes place at a certain period of
time
The reactants are combined

The reaction proceeds for a designated

time

The reaction is stopped & measurement

is made.
2. Continuous monitoring/ Kinetic Assay
Measured absorbances at a certain
time period
Multiple measurements of absorbance
changed are made during the
reaction;
MORE ADVANTAGEOUS than Fixed-
time because it allows to monitor
linearity.
 Units for expressing Enzymatic
Activity
1. International Unit (IU/ U)
 Conventional unit
 Amount of enzyme that catalyzes 1 micromole of
substrate/ minute.
2. Katal Unit (KU)
 1 mole of substrate/seconds

1 IU=16.7 nkat/ 17 nkat, 1 kat/L= 0.06 IU/L

NTR:
 Enzymes are quantitated based on their activity rather
than absolute values.
 The units used to report enzyme levels are ACTIVITY
UNITS.
 The definition for activity unit must consider change in
pH, temp., substrate, etc.
 THE END 
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