Notes in Enzymology Prepared by Mark Rodrigo D.

Mendros

CLINICAL CHEMISTRY 2

 Enzyme
 Biologic protein that catalyze biochemical reactions
 ___________________________________________
 ___________________________________________
 Functions of Enzymes
 ____________________________
 ____________________________
 ____________________________
 ____________________________
 ____________________________
 ____________________________
 General Properties and Definitions
 Components of an Enzyme
 Active Site:
________________________________________________________________________
__________________________________________________________ _____________
 Allosteric Site :
________________________________________________________________________
________________________________________________________________________

 Terms associated with enzymes

 Substrates : ______________________________________________________________
 Cofactors : ______________________________________________________________
 Coenzyme : __________________________________________________________
 Activator : ___________________________________________________________
 Isoenzyme: ___________________________________________________________
 Apoenzyme: __________________________________________________________
 Holoenzyme: _________________________________________________________

 Enzyme Classification and Nomenclature

 Enzyme Commission of IUB
 Systematic name: __________________________________________________
 Recommended name: _______________________________________________
 EC numerical code : _________________________________________________
o 4 digits

1. Oxidoreductases
 A- + B → A + B-
 E.g:_______________________________________________
2. Transferases
 A-X + B → A + B-X
 E.g: ______________________________________________
3. Hydrolases
 A–B + H2O → A–OH + B–H
 E.g. : _____________________________________________
4. Lyases
 ATP → cAMP + PPi
 Notes in Enzymology Prepared by Mark Rodrigo D. Mendros

 E.g. : _____________________________________________
5. Isomerases
 A→B
 E.g.: _____________________________________________________
6. Ligases
 Ab + C → A–C + b
 E.g. : _____________________________________________________

 Enzyme Kinetics
 Catalytic Mechanism of Enzymes
 Enzymes catalyze physiologic reactions by lowering the activation energy level that the
reactants must reach

 Catalytic Mechanism of Enzymes

 Absolute specificity : _____________________________________________________
 Group specificity: ________________________________________________________
 Bond specificity: _________________________________________________________
 Stereoisometric specificity: _________________________________________________
 Factors that Influence Enzymatic Reactions
 Substrate Concentration:
________________________________________________________________________
________________________________________________________________________
 Enzyme Concentration:
________________________________________________________________________
________________________________________________________________________
 pH : ____________________________________________________________________
 Temperature: ____________________________________________________________
 Cofactors: _______________________________________________________________
 Inhibitors: _______________________________________________________________
 3 types: __________________________________________________________

 Measurement of catalytic activity

 Increase in product concentration
 Decrease in substrate concentration
 Decrease in coenzyme concentration (NADH)
 Increase in altered coenzyme concentration
 Notes in Enzymology Prepared by Mark Rodrigo D. Mendros

 Measurement of catalytic activity

 Dependent on enzyme concentration
 Always performed in zero-order kinetics
 Performed during the linear phase of reaction
 General methods of measuring enzymatic reaction
 Fixed-time
________________________________________________________________________
________________________________________________________________________
 Continuous monitoring
________________________________________________________________________
________________________________________________________________________

 Calculation of Enzyme Activity

 IU (EC)
 the amount of enzyme that will catalyze the reaction of 1 μmol of substrate per
minute.
 Kat (SI)
 the amount of enzyme that will catalyze the reaction of 1 mol of substrate per
second.
 Enzyme Theories
 Emil Fisher's/ Lock and Key theory :
________________________________________________________________________
 Kochland's Induced Fit Theory :
________________________________________________________________________

Enzymes of Clinical Significance

 LIVER ENZYMES
1. Aspartate Aminotransferase (AST)
 Serum glutamic-oxaloacetic transaminase (SGOT)
 Transfer of an amino group between aspartate and α-keto acids
 Involved in the synthesis and degradation of AA.
 Widely distributed, highest activities in cardiac, liver and skeletal muscle.
 2 Isoenzymes: _______________________________________________

 Diagnostic Significance
• After MI, AST levels begin to rise in 6-8 hours, peak at 24 hours, and
return to normal in 5 days.
• Also ↑ in hepatocellular and skeletal muscle dis.

 Assay for Enzyme Activity

 Karmen Method
 Uses malate dehydrogenase and monitors decrease in absorbance at
340 nm
 Falsely ↑ in hemolyzed sample
 Reference Range: 5 – 30 U/L
 Notes in Enzymology Prepared by Mark Rodrigo D. Mendros

2. Alanine Aminotransferase (ALT)

 Also known as serum glutamic-pyruvic transaminase (SGPT)
 Transfer of an amino group between alanine and α-ketoglutarate
 Increased in hepatocellular disorders.

 De Ritis Ratio
 The AST/ALT Ratio
 Differentiates the cause of hepatic disorder
 Ratio > 1  __________________
 Ratio < 1  __________________
 Assay for Enzyme Activity
 Uses Lactate Dehydrogenase and monitors decrease in absorbance at
340 nm
 Reference Range: 6-37 U/L

SGOT/AST SGPT/ALT

MAJOR ORGAN AFFECTED

SUBSTRATE

END PRODUCTS

COLOR DEVELOPER

COLOR INTENSIFIER
 Notes in Enzymology Prepared by Mark Rodrigo D. Mendros

METHODS

3. Gamma-Glutamyltransferase (GGT)
 Catalyze the transfer of the γ-glutamyl residue from γ-glutamyl peptides to amino acids, H20,
etc.
 Used for diagnosis hepatobiliary disorders and chronic alcoholism

 Assay for Enzyme Activity

 Szaz Assay
 The absorbance of p-Nitroaniline is measured at 405-420 nm

 M.I. Profile
4.Creatine Kinase (CK)
 Involved in the storage of high-energy creatine phosphate in muscle cells
 Widely distributed, highest activities in skeletal muscle, heart, and brain

 Methods of Determination
i. Forward Reaction (Tanzer-Gilvarg)
• ↓ in absorbance at 340 nm is determined
• Optimum pH is 9.0

ii. Reverse Reaction (Oliver-Rosalki)

• ↑ in absorbance at 340 nm is determined
• Optimum pH: 6.8
 Notes in Enzymology Prepared by Mark Rodrigo D. Mendros

 Source of Error
o Hemolysis cause false ↑ CK activity
o CK is inactivated by light
o Physical activity and IM injections cause ↑ CK
o Reference Range
 M  15-160 U/L : F  15-130 U/L
 CK-MB: <6% of total CK
Ck-3 / CK-MM / Muscle type CK- 2 / CK-MB / Hybrid Type CK-1 / CK-BB / Brain Type

o Diagnostic Significance of CK Isoenzymes

 After MI, CK-MB (>6%) levels begin to rise within 4-8 hours, peak at 12-24 hours, and
returns to normal levels within 48-72 hours.

o Other CK Isoenzymes
 Macro-CK
 ____________________________________
 Mitochondrial CK (CK-Mi)
 ____________________________________

5.Lactate Dehydrogenase (LD)

 Catalyzes the interconversion of lactic and pyruvic acids
 Widely distributed, highest activities in heart, hepatic, skeletal muscle and RBC

 Assay of Enzyme Activity

 Wacker method
 Forward Reaction (Lactate  Pyruvate)
 Notes in Enzymology Prepared by Mark Rodrigo D. Mendros

 Increase in absorbance is monitored at 340 nm

 Optimal pH is 8.3 – 8.9

 Wrobleuski La Due
 Reverse Reaction (Pyruvate  Lactate)
 Decrease in absorbance is monitored at 340 nm
 Three times faster but more susceptible to substrate exhaustion
 Optimal pH is 7.1 to 7.4

 Assay of Enzyme Activity

 α-hydroxybutyrate dehydrogenase (α-HBD)
 Has greater affinity of H subunits
 Represent LDH-1

 After MI, LD begin to rise within 10-24 hours, peak at 48-72 hours, and remains elevated for 10 days.
 Reference Range: 100-225 U/L

Lactate Dehydrogenase (LDH) Isoenzyme

CK-MB AST LDH

 Notes in Enzymology Prepared by Mark Rodrigo D. Mendros

Appearance

Peak

Stay Elevated

 Other Liver Enzymes

6. Alkaline Phosphatase
 Catalyze the hydrolysis of various phosphomonoesters at an alkaline pH
 Liberate inorganic phosphate from an organic phosphate ester with production
of alcohol
 Requires Mg2+ activator
 For evaluation of hepatobiliary and bone disorders.

1. Assay for Enzyme Activity

 Bowers and McComb
 Based on molar absorptivity of p-Nitrophenol
 Absorbance is measured at 405 nm

Other methods:

Methods Substrate End Product

(1-4) Bodansky, Shinowara, Inorganic PO4 +

β-glycero-phosphate
Jones, Reinhart Glycerol

(5) Bessy, Lowry & Brock p-nitrophenyl phosphate p-nitrophenol (yellow)

(6) Bowers & McComb

(7) King and Armstrong Phenyl phosphate phenol

 Reference Range
 30 – 90 U/L (Adult)
 Notes in Enzymology Prepared by Mark Rodrigo D. Mendros

 70 – 220 U/L (0 – 3 months)

 50 – 260 U/L (3 - 10 years)
 60 – 295 U/L (10 - puberty)

 ALP Isoenzymes
 Differentiation by electrophoresis
 Liver ALP: _____________________________________________
 Bone ALP: _____________________________________________
 Placental ALP: __________________________________________
 Intestinal ALP: __________________________________________

 CARCINOPLACENTAL ALP
1. REGAN ALP : _________________________________________________________
2. NAGAO ALP : _________________________________________________________

7. Acid Phosphatase (ACP)

 Catalyze the hydrolysis of various phosphomonoesters at an acid pH
 Liberate inorganic phosphate from an organic phosphate ester with production
of alcohol
 For evaluation of metastatic carcinoma of prostate.
 Forensic investigation of rape

 To differentiate the prostatic form from the non-specific form like RBC acid
Phosphatase inhibitors are added
 L-tartrate ions – __________________________________
 Formaldehyde and Cupric ions – _____________________

 Assay for Enzyme Activity

 Reference Range: Prostatic ACP: 0 -3.5 ng/ml

Methods Substrate

(1) Quantitative end point

(2) Continuous monitoring

 Notes in Enzymology Prepared by Mark Rodrigo D. Mendros

METHODS SUBSTRATE END PRODUCTS

GUTMAN AND GUTMAN PHENYL PO4 INORGANIC PHOSPHATE

SHINOWARA PNPP P-NITROPHENOL

BABSON, READ AND PHILLIPS ALPHA NAPHTYL ALPHA NAPHTOL

PO4

ROY AND THYMOLPHTHALEIN FREE THYMOLPHTHALEIN

HILLMAN MonoPO4

 Pancreatic Enzymes
1. Amylase (AMS)
 Catalyzes the breakdown of starch and glycogen via α, 1-6 branching linkages
 Increased in acute pancreatitis, renal failure and parotitis

 Amylase Isoenzymes
 Salivary Amylase  __________________________
 Pancreatic Amylase  ________________________

Amylase Methodologies

Amyloclastic Measures the disappearance of starch substrate

Starch-Iodine Complex (Dark-blue) 

Saccharogenic Measures the appearance of the product

Starch 

Amylase Methodologies

Chromogenic Measures the increasing color from production of product -

chromogenic dye fragment

Insoluble starch-dye  soluble starch-dye fragments

 Notes in Enzymology Prepared by Mark Rodrigo D. Mendros

Continuous monitoring Coupling of several enzyme systems to monitor amylase activity

2. Lipase (LPS)
o Hydrolyzes the ester linkages of fats to produce alcohols and fatty acids
o Hydrolysis of dietary triglycerides in the intestine to 2-monoglyceride and fatty acids

o Assay for Enzyme Activity

Cherry Crandall Tietz

Substrate

Titrating agent

Indicator

Endpoint

End Color

o Turbidimetric methods
 Estimation of liberated fatty acids
Notes in Enzymology Prepared by Mark Rodrigo D. Mendros