ENZYMES

• Produced by living cells; proteins in nature

• Measured in terms of their activity and not in terms of
their absolute values.

Factors Affecting Enzymatic Reactions:

1. Enzyme Concentration
2. Substrate Concentration
3. Cofactors
a. Coezymes - NADP, NAD
b. Activators – Ca, Mg, Zn, Cl, K
c. Metalloenzymes – catalase and CO
4. Inhibitors
a. Competitive inhibitor (reversible)
• Binds to the active site of the enzyme.
• Substrate + inhibitor competes for the
same active site.
• Remedy: ↑ substrate + serum dilution

b. Non-competitive inhibitor (irreversible)

• Inhibitor binds at a place other than the
active site.
• Substrate + inhibitor do not compete.
c. Uncompetitive inhibitor
• Inhibitor binds to the ES complex.
• ↑ substrate = ↑ inhibition
Michaelis Menten Kinetics
Enzyme + Substrate ES complex

Product + Free Enzyme

4
2 Enzymatic Theories:
1. Emil Fisher’s Theory (Lock and Key)
• The shape of the key (substrate) must
conform into the lock (enzyme).

2. Kochland’s Theory (Induced Fit)

• Based on the attachment of a substrate to
the active site of the enzyme.

5
2 Enzymatic Reactions:
1. Zero Order reaction
2. First Order reaction

2 Ways of Expressing Enzymatic Activity:

1. International Unit (IU/U)
• Amount of enzyme that catalyzes one micromole of
substrate/minute.
2. Katal Unit (KU)
• Amount of enzyme that catalyzes one mole of
substrate/second.

• 1 IU = 16.7nkat
• 1kat/L = 0.06 U/L
6
Classification of Enzymes:
1.Oxidoreductases
2.Transferases
3.Hydrolases
4.Lyases
5.Isomerases
6.Ligases
ALKALINE PHOSPHATASE
• Hepatic and bone marker
Isoenzymes:
• Liver ALP
• Bone ALP - heat labile
• Placental ALP - heat stable
• Intestinal ALP
Methods: (substrate - PNPP)
1. Bessy, Lowry and Brock
2. Bowers and Comb
Carcinoplacental ALP:
1. Regan ALP – most heat stable (65°C- 30mins)
2. Nagao ALP – inhibited by L- leucine
8
 TRANSAMINASES
• Significant in the evaluation of MI, hepatocellular
disorders and skeletal muscle disorders.

SGOT - cardiac tissue, liver and skeletal muscle

- kidney, pancreas and RBC (small amt)
• In AMI, AST rises at 6-8 hrs., peaks at 24 hrs., normalize
w/in 5 days.

SGPT - liver (highest level); specific test

- kidney, skeletal muscles, cardiac tissue
& spleen, RBC (small amt)

• Cofactor: Pyridoxal phosphate (vitamin B6)

• Methods: Reitman and Frankel; Karmen
9
ACID PHOSPHATASE
• For detection of prostatic CA; forensic
chemistry.
• Found in prostate, RBC, platelets, bone,
spleen and kidneys.
• Method: Roy and Hillman (thymolphthalein
monoPO4)
• At RT, decreases w/in 1-2 hours (citric acid
is added as stabilizer)
10
Prostatic ACP Red Cell/Non Prostatic ACP

Specific ACP Non-specific ACP

Inhibited by: Inhibited by: HCHO

L-tartrate & cupric ions

11
AMYLASE
• Catalyzes the breakdown of starch.
• Organ affected: Pancreas
• Isoenzymes: Salivary AMS – anodal
Pancreatic AMS – cathodal
• The smallest enzyme in size (50,000 MW) – readily
filtered by the glomerulus and appears in urine.
In acute pancreatitis:
Initial rise: 2-12 hours after onset of an
attack (earliest marker)
Peak level: 24 hours
Normal level: 3-5 days

Methods: (substrate – starch)

• Saccharogenic, Amyloclastic, Chronometric 12
LIPASE
• An enzyme that catalyzes the ester linkages of fats to
produce alcohol and FA.
• Organ affected: Pancreas
• Specific marker for acute pancreatitis - not affected
by renal disorder.
In acute pancreatitis:
Initial rise: 6 hours after onset of attack
Peak: 24 hours
Elevated: 7 days (late pancreatic marker)

Methods: Cherry Crandall (substrate – 50% olive oil or

triolein)
13
LACTIC DEHYDROGENASE
• Useful in monitoring myocardial, hematological
and hepatic disorders.
• Lacks specificity due to various organ sources.

LD1 & LD2 - heart muscles, RBC & renal cortex

LD3 - lungs, spleen, pancreas &
WBC (lymphocytes)
LD4 & LD5 - liver, skeletal muscles, ileum
and skin

14
• Physiologically, LD2 > LD1

“Flipped Pattern” = LD1 > LD2

1. myocardial infarction
2. hemolytic anemia
3. renal infarction
• The highest elevation is in pernicious anemia
and hemolytic disorders.

15
• LD-6 = alcohol dehydrogenase
(arteriosclerotic failure)
Methods:
1. Wacker Method (Forward/Direct Rxn)
2. Wrobleuski La Due (Reverse/Indirect)

16
CREATINE PHOSPHOKINASE (CPK)
• Detects damages to myocardial and skeletal
muscles - AMI and Duchenne disorder (CK-
MM).
• Duchenne disorder – highest elevation of CK

• CPK 1 or BB - brain, lungs, prostate,

kidney, thyroid, uterus and intestine
• CPK 2 or MB - heart muscles, tongue,
esophagus
• CPK 3 or MM – heart and skeletal muscles 17
NTR:
• CK-BB rarely seen in serum of adults (↑ high
molecular size
• CK-MM is the major isoenzyme in the normal
sera (94-100%).
• Sudden appearance in serum suggests
myocardial release = CK-MB > 6% of the total
CPK (AMI).
• Liver cells and RBC do not contain CK.
Methods:
1. Tanzer-Gilbarg (forward/direct rxn)
2. Rosalki and Hess (reverse/indirect) 18