Introduction to ENZYMES

Enzymes

• Proteins
• Measured in terms of their activity
• Abnormality= organ damage
• Each enzyme consist of an active site
– water-free cavity, where the substance on which enzyme acts
(substrate) interacts with particular charged amino acid residues;
3D
• Allosteric site
– a cavity other than the active site—may bind regulator molecules
and, thereby, be significant to the basic enzyme structure.
ENZYME CLASSIFICATION AND NOMENCLATURE

• Enzyme Commission (EC) of the Internation Union of

Biochemistry (IUB) on 1961= adapted the systematic name
to each enzyme defining the:
– substrate acted on
– reaction catalyzed
– name of any coenzyme involved in the reaction

EC NUMERICAL CODE (4 DIGITS):

– 1st digit = enzyme classes
– 2 and 3rd = subclass and sub-subclass of the enzyme
– Final number = serial number specific to each enzyme in a subclass
Class Function Example
1. Oxidoreductases Catalyze an oxidation–reduction reaction Cytochrome oxidase, LD, G6PD,
between two substrates Malate dehydrogenase, isocitrate
dehydrogenase
2. Transferases Catalyze the transfer of a group other than CK, AST, ALT
hydrogen from one substrate to another

3. Hydrolases Catalyze hydrolysis of various bonds Esterases = ACP, ALP, Chalcone

4. Lyases Catalyze removal of groups from substrates
without hydrolysis; the product contains double
bonds
5. Isomerases Catalyze the interconversion of geometric,
optical, or positional isomers
syntahase, LPS
Peptidases= Trypsin, Pepsin, LAP
Glycosidase= AMS, Galactosidase
Glutamate decarboxylase,
pyruvate decarboxylase,
tryptophan decarboxylase, and
aldolase
Glucose phosphate isomerase,
ribose phosphate isomerase

6. Ligases Catalyze the joining of two substrate molecules, Synthase

coupled with breaking of the pyrophosphate
bond in adenosine triphosphate (ATP) or a
similar compound
Enzyme Theory

1. Emil Fisher’s/ Lock and Key Theory

✓ Based o the premise that the shape of the key (substrate) must fit into the
lock (enzyme)
2. Kochland’s/Induced Fit Theory
✓ Based on the substrate binding to the active site of the enzyme
ENZYME KINETICS

• Activation energy
– excess energy and the energy required to raise all molecules in 1
mole of a compound at a certain temperature to the transition
state at the peak of the energy barrier
• Absolute specifity
– enzyme combines with only 1 substrate and catalyzes only 1 reaction
• Group specificity
– enzymes combine with all the substrates in a chemical group
• Bond specificity
– enzymes reacting with specific bonds
• Enzymes catalyze physiologic reactions by lowering the
activation energy level that the reactants (substrates)
must reach for the reaction to occur
• General relationship among enzyme, substrate, and
product may be represented as follows:
• E + S --> ES --> E + P
ENZYME ACTIVITY

• First-order Kinetics/reactions
✓ reaction rate is directly proportional to substrate concentration
✓ the reaction rate steadily increases when more substrate is
added, with the amount of enzyme exceeding the amount of
substrate
• Zero-order kinetics/reactions
✓ reaction rate depends only on enzyme concentration
✓ always performed for the measurement of enzyme
concentration
ENZYME ACTIVITY
• To measure the extent of enzymatic reactions, 2 general methods may be used:
✓ Fixed-time - the reactants are combined; the reaction proceeds for a designated time; the
reaction is stopped and measurement is made
✓ The reaction is assumed to be linear over the reaction time; the larger the reaction, the more enzyme
is present.
✓ Continuous monitoring/ kinetic assay - multiple measurements of changed in absorbance are
made during the reaction (either time intervals or continuously) using a continuous recording
spectrophotometer;
✓ preffered test = the linearity of the reaction may be more adequately verified. If absorbance is
measured at intervals several data points are necessary to increase the accuracy of linearity
assessment; any deviation from linearity is readily observable.
• Enzymes are measured in terms of:
➢ Change in the substrate concentration
➢ Change in the product concentration
➢ Change in coenzyme concentration
Units of Expressing Enzymatic Activity

1. International Unit (IU or U) - 1 micromole of substrate/

minute
2. Katal Unit (KU) - 1 mole of substrate/second
Factors that Influence Enzymatic Reactions
1. ENZYME CONCENTRATION
2. COFACTORS
✓ nonprotein entities that must bind to particular enzymes before a
reaction occurs; basic enzyme structure
✓ Coenzymes- organic cofactors that is essential to achieve absolute enzymatic
activity; second subtrate (ex. NADH)
✓Prosthetic group= tightly bound to the enzyme
✓Apoenzyme= enzyme portion
✓Holoenzyme= complete and active system (apoenz + prosthetic grp)
✓Proenzyme/zymogen= inactive form
✓ Activators- inorganic cofactors that alters the spatial configuration of the
enzymes for proper subtrate binding
✓ Metalloenzymes- inorganic compound attached to a molecule
Factors that Influence Enzymatic Reactions
3. SUBSTRATE CONCENTRATION
✓ One major influence on enzymatic reactions
✓ 1913- Michaelis and Menten hypothesized the Enzyme-Substrate (ES)
complex
✓ Michaeles-Menten constant (Km) = a constant for a specific
enzyme and substrate under defined conditions; expression of the
relationship between the velocity of an enzymatic reaction and
substrate concentration; indicates the amount of substrate needed
for a particular enzymatic reaction
Factors that Influence Enzymatic Reactions
4. INHIBITORS
✓ Enzymatic reactions may not progress if an inhibitor interferes with
the reaction

Types:
a) Competetive Inhibitor
▪ physically binds to the active site of an enzyme
▪ both the substrate and inhibitor compete for the same active site of the enzyme
b) Non-competetive
▪ does not compete with the substrate but look for areas other than the active site
c) Uncompetetive
▪ the inhibitor binds to the enzyme-substrate (ES) complex
Factors that Influence Enzymatic Reactions
5. ISOENZYMES
✓ enzymes having the same catalytic reactions but slightly different molecular structures

6. HYDROGEN ION CONCENTRATION (pH)

✓ pH 7 to 8
✓ Extreme pH= denatures enzyme

7. HEMOLYSIS
✓ mostly increases enzyme reaction

8. LACTESCENSE OR MILKY SPECIMEN

✓ decreases enzyme concentration
Factors that Influence Enzymatic Reactions
9. TEMPERATURE
✓ Enzymes are active at: 25°C, 30°C, 37°C (optimum temp)
✓ 40°C to 50°C = increases denaturation of enzymes
✓ 60°C to 65°C = inactivation of enzymes
✓ Temperature Coefficient (Q10): every 10°C in temp = 2-fold inc in enz activity

10. STORAGE
✓ Low temperatures (refrigeration/freezing = reversibly active enzyme
✓ Repeated freezing and thawing = denature proteins
✓ -20°C = preservation for longer period of time (enzyme)
✓ Room temperature = ideal storage of LDH (LD4 and LD5)
Causes of Elevated Plasma Enzyme level

1. Impaired removal of enzyme from plasma

2. Increased permeability of cell membrane
3. Increased in the number of cells or the production of cells
4. Increased in the normal cell turnover
5. Decreased clearance of enzymes from the circulation
6. Tissue necrosis and degeneration