ENZYMOLOGY PART 1 o “even in low substrate concentration, the
substrate can readily bind with free enzyme”
ENZYMES
ESSENTIAL TO PHYSIOLOGIC FUNCTIONING:
o Hydration of CO2
o Nerve conduction
o Muscle contraction
o Nutrient degradation
o Energy use
o Present inside the cell
o Speeds up biochemical reactions
o Reactants:
o Enzyme
 Active Site - the area where the
substrate binds to the enzyme
 Allosteric Site - primarily made
up of water
o Substrate
o Forms the ES complex
 Fast chemical reaction
CATALYTIC MECHANISM OF ENZYMES
o A chemical reaction may occur spontaneously if the
free energy or available kinetic energy is higher for
the reactant than the products (lower energy).
o Activation Energy  reactants have enough energy
to break their bond and collide to form new bond
ENZYMES ARE HIGHLY SPECIFIC:
o Absolute Specificity  the enzyme combines with
only one substrate and catalyze a single reaction
(strictest)
o Group Specificity  the enzyme combines with all
substrates with a particular chemical group
o Bond Specificity  the enzyme combines with all
substrates with a particular chemical bond
o Stereoisometric Specificity  the enzyme combines
with all substrate with a specific optical isomer
FACTORS THAT INFLUENCE ENZYMATIC REACTION
1. SUBSTRATE CONCENTRATION/ENZYME
CONCENTRATION
o Follows the hypothesis of Michaelis & Menten 
o Enzymatic Reaction can be:
 First-Order Kinetic : the rate of reaction is
dependent to the substrate concentration
 Zero-order kinetic : the rate of reaction is
dependent to the enzyme concentration
2. PH -
o Enzymes are protein that carry a net molecular
charge
o Optimal pH for enzyme reaction is pH of 7-8
3. TEMPERATURE
o Temperature coefficient - in every 10 °C increase
in the temperature, it will result to two-folds
increase in the activity of enzyme
o 40-50*C  denaturation of enzyme
o 60-65*C  inactivation of enzyme
o 378C (25 or 30*C)  optimum temperature for
enzyme reaction
4. CO-FACTORS - non-protein entities that must bind
to an enzyme for a reaction to occur
o Activators  inorganic cofactors
 metallic Ca, Fe, Mg, Mn, Zn, K
 non-metallic Br, Cl
 alter the spatial configuration of enzyme
o Coenzyme  organic cofactors
o serve as a second substrate to enzyme reaction
o e.g. vitamins, NAD, nucleotide phosphatase
5. INHIBITORS - interferes with the enzyme reaction
o Types of Inhibition
● Competitive Inhibition – targets the
active site
● Non-Competitive Inhibition - targets the
allosteric site
● Uncompetitive Inhibition - targets the
ES complex
6. STORAGE
o 20*C  long term preservation
o 2-8*C  general temperature for preservation
(most used)
o RT  for preservation of cold labile enzymes
(Ex. LD4 & LD5)
o Cold temperature - can cause reversible
inactivation of enzymes.
o Repeated thawing - causes inactivation of
enzyme
 NOMENCLATURE
7. HEMOLYSIS
Enzymes that are affected: Developed by the E.C. (Enzyme Commission)

K - Potassium 1. Oxido-reductase - catalyze an oxidation-reduction

L - Lactate dehydrogenase reaction between two substrates
A - Acid phosphatase, aspartate transferase,
aldolase 2. Transferase - catalyze the transfer of a group other
M - Magnesium than hydrogen from one substrate to another
P - Phosphorus
3. Hydrolase - catalyze hydrolysis of various bonds
8. LACTESCENCE/MILKY SPECIMEN – decrease in
4. Lyase - catalyze removal of groups from substrates
concentration (caused by chylomicrons)
without hydrolysis; the product contains double
ENZYMATIC REACTIONS bonds

● measures enzyme based on their activity not by

5. Isomerase - catalyze the interconversion of
their absolute value
geometric, optical, or positional isomers
2 Methods in measuring the enzymatic reactions:

o Fixed time (end point)  Single measurement of 6. Ligases - catalyze the joining of two substrate
enzyme activity molecules, coupled with breaking of the
o Continuous-Monitoring/Kinetic Assay  Multiple pyrophosphate bond in adenosine triphosphate
measurement of enzyme activity (ATP) or a similar compound

ENZYMES IS A PROTEIN (AMINO ACID)

o Primary Structure  specific sequence of amino acid

o Secondary Structure  polypeptide chain twist
o Tertiary structure  folding of the polypeptide
chains
o Quaternary structure  combination of tertiary
structure

ISOENZYMES

● enzymes with same catalytic function but

different physical properties
● CK has 3 isoenzyme
● CK1 –CKBB (Brain)
● CK2
● CK3 – CKMM (Muscle)
Differentiated by:

o Electrophoretic mobility
o Solubility
o Resistance to inactivation