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Enzymes
I. Introduction
I. Introduction
Enzyme
I. Introduction
Function of Enzymes
II. General Properties and Definitions
II. General Properties and Definitions
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II. General Properties and Definitions II. General Properties and Definitions
II. General Properties and Definitions

II. General Properties and Definitions

II. General Properties and Definitions
co-enzyme
apoenzyme
III. Enzyme Classification and Nomenclature
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III. Enzyme Classification and Nomenclature III. Enzyme Classification and Nomenclature

III. Enzyme Classification and Nomenclature

III. Enzyme Classification and Nomenclature

IV. Enzyme Kinetics
IV. Enzyme Kinetics
i. Catalytic Mechanism of Enzymes
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IV. Enzyme Kinetics IV. Enzyme Kinetics
IV. Enzyme Kinetics
IV. Enzyme Kinetics
ii. Factors that Influence Enzymatic Reactions
IV. Enzyme Kinetics
IV. Enzyme Kinetics
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IV. Enzyme Kinetics
IV. Enzyme Kinetics
IV. Enzyme Kinetics IV. Enzyme Kinetics

IV. Enzyme Kinetics

IV. Enzyme Kinetics
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IV. Enzyme Kinetics
iii. Measurement of Enzyme Activity
IV. Enzyme Kinetics
iii. Measurement of Enzyme Activity
IV. Enzyme Kinetics
iii. Measurement of Enzyme Activity
IV. Enzyme Kinetics
iii. Measurement of Enzyme Activity
IV. Enzyme Kinetics
iii. Measurement of Enzyme Activity
IV. Enzyme Kinetics
iv. Calculation of Enzyme Activity
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IV. Enzyme Kinetics
iv. Calculation of Enzyme Activity
IV. Enzyme Kinetics
v. Measurement of Enzyme Mass
IV. Enzyme Kinetics
v. Measurement of Enzyme Mass
IV. Enzyme Kinetics
V. Enzymes of Clinical Significance
V. Enzymes of Clinical Significance
C. Pancreatic Enymes
1. AMS
2. LPS
D. Other Enzymes
1. ACP
2. G-6-PDH
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V. Enzymes of Clinical Significance
A. MI Profile
1. Creatinine Kinase (CK)
V. Enzymes of Clinical Significance
V. Enzymes of Clinical Significance
in absorbance at 340 nm is determined
Optimum pH is 9.0
V. Enzymes of Clinical Significance
in absorbance at 340 nm is determined
Optimum pH: 6.8
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V. Enzymes of Clinical Significance
:
V. Enzymes of Clinical Significance
V. Enzymes of Clinical Significance
Ck-3 / CK-MM / Muscle
type
CK- 2 / CK-MB /
Hybrid Type
CK-1 / CK-BB /
Brain Type
Slowest mobility
toward the anode
2
nd
fastest to migrate
toward the anode
Migrate fastest
toward the anode
Major isoenzyme in
striated muscle and
normal serum
Significant quantities
are found in heart
tissues
Highest concentration
in CNS, GI tract and
uterus (pregnancy)
V. Enzymes of Clinical Significance
V. Enzymes of Clinical Significance V. Enzymes of Clinical Significance
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V. Enzymes of Clinical Significance V. Enzymes of Clinical Significance
V. Enzymes of Clinical Significance V. Enzymes of Clinical Significance
V. Enzymes of Clinical Significance
CK-MB (>6%)
4-8 hours 12-24 hours
48-72 hours
V. Enzymes of Clinical Significance
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Reference Values:
94-98% CK-MM
2-6% CK-MB
Anode (+)
Cathode (-)
Separation of CK isoenzymes by
electrophoresis
NC MI
V. Enzymes of Clinical Significance
A. MI Profile
2. Aspartate Aminotransferase (AST)
V. Enzymes of Clinical Significance
V. Enzymes of Clinical Significance
AST 6-8 hours
24 hours 5 days
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V. Enzymes of Clinical Significance
Karmen Method

V. Enzymes of Clinical Significance

A. MI Profile
3. Lactate Dehydrogenase (LD)
V. Enzymes of Clinical Significance
V. Enzymes of Clinical Significance
V. Enzymes of Clinical Significance
LD 12-24 hours
48-72 hours 10
days
:
V. Enzymes of Clinical Significance
Wacker method

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V. Enzymes of Clinical Significance
Wrobleuski La Due

V. Enzymes of Clinical Significance

-hydroxybutyrate dehydrogenase (-HBD)
V. Enzymes of Clinical Significance
Isoenzyme Tissue Disorder ()
LDH-1 (HHHH) Heart, RBC MI, Hemolytic anemia
LDH-2 (HHHM) Heart, RBC RI, Megaloblastic anemia
LDH-3 (HHMM) Lung, Spleen, Pancreas Pulmonary embolism
LDH-4 (HMMM) Liver Hepatic injury
LDH-5 (MMMM) Skeletal Muscle Skeletal muscle injury
Lactate Dehydrogenase (LDH) Isoenzyme
V. Enzymes of Clinical Significance
> > >
>
AMI Intravascular hemolysis
Flipped pattern >
V. Enzymes of Clinical Significance
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Correspondence Between CPK and LDH Isoenzyme Patterns
V. Enzymes of Clinical Significance
A. MI Profile
CK-MB AST LDH
Appearance 4-8 hours 6-8 hours 10-24 hours
Peak 12-24 hours 24 hours 48-72 hours
Stay
Elevated
3 days 5 days 10 days
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V. Enzymes of Clinical Significance
A. MI Profile
1. Alanine Aminotransferase (ALT)
V. Enzymes of Clinical Significance
V. Enzymes of Clinical Significance

V. Enzymes of Clinical Significance

V. Enzymes of Clinical Significance

A. MI Profile
2. Alkaline Phosphatase (ALP)
V. Enzymes of Clinical Significance
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V. Enzymes of Clinical Significance
Bowers and McComb

V. Enzymes of Clinical Significance

Methods Substrate End Product
(1-4) Bodansky, Shinowara,
Jones, Reinhart
-glycero-
phosphate
Inorganic PO
4
+ Glycerol
(5) Bessy, Lowry & Brock
(6) Bowers & McComb
p-nitrophenyl
phosphate
p-nitrophenol
(yellow)
(7) King and Armstrong Phenyl phosphate phenol
V. Enzymes of Clinical Significance
V. Enzymes of Clinical Significance V. Enzymes of Clinical Significance
Fastest isoenzyme and in liver diseases
Two fractions: Major liver & fast liver (
1
) band
Heat labile fraction
in bone disease, healing of bone fractures and
physiologic bone growth
Pagets disease (osteitis deformans)
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V. Enzymes of Clinical Significance
Most heat stable fraction and in pregnancy
16-20 weeks / complications of pregnancy
Slowest moving fraction, in blood groups B or O
in fatty meal consumption and GIT disorders
Note: Placental and Intestinal ALP are inhibited
by phenylalanine (chemical inhibition)
V. Enzymes of Clinical Significance
Differentiation by Heat Stability
1. Liver ALP
ALP residual activity is to >20%
2. Bone ALP
ALP residual activity is to <20%
V. Enzymes of Clinical Significance
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V. Enzymes of Clinical Significance
A. MI Profile
4. Gamma glutamyl transferase (GGTP)
V. Enzymes of Clinical Significance V. Enzymes of Clinical Significance
Szaz Assay
V. Enzymes of Clinical Significance
A. MI Profile
V. Enzymes of Clinical Significance
1. Amylase (AMS)
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V. Enzymes of Clinical Significance V. Enzymes of Clinical Significance

V. Enzymes of Clinical Significance

Amylase Methodologies
Amyloclastic Measures the disappearance of starch substrate
Starch-Iodine Complex (Dark-blue)
Decrease in color intensity
Saccharogenic Measures the appearance of the product
Starch reducing sugars
V. Enzymes of Clinical Significance
Amylase Methodologies
Chromogenic Measures the increasing color fromproduction of
product -chromogenic dye fragment
Insoluble starch-dye soluble starch-dye
fragments
Continuous
monitoring
Coupling of several enzyme systems to monitor
amylase activity
V. Enzymes of Clinical Significance V. Enzymes of Clinical Significance
2. Lipase (LPS)
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V. Enzymes of Clinical Significance V. Enzymes of Clinical Significance
CherryCrandall Tietz
Substrate 50%olive oil (triolein)
Titrating agent 0.4N NaOH
Indicator
Phenolpthalein
Thymolpthalein +
Veronal
Endpoint Fatty Acid (Oliec Acid)
End Color Pink Blue
V. Enzymes of Clinical Significance
Estimation of liberated fatty acids
V. Enzymes of Clinical Significance
1. Acid Phosphatase (ACP)
V. Enzymes of Clinical Significance
metastatic carcinoma of prostate
V. Enzymes of Clinical Significance
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V. Enzymes of Clinical Significance
Methods Substrate
(1) Quantitative end point Thymolpthalein monophosphate
(2) Continuous monitoring -napthyl phosphate
V. Enzymes of Clinical Significance
1. Acid Phosphatase (ACP)
V. Enzymes of Clinical Significance
C. Pancreatic Enymes
1. AMS
2. LPS
D. Other Enzymes
1. ACP
2. G-6-PDH