enzymology

1.  An enzyme is a protein that catalyzes one or
more specific biochemical reactions without
altering the equilibrium point of reaction or being
consumed or changed in a reaction.
 Enzymes are present in high concentration in
cells as compared to plasma.
 Enzyme activity in plasma:
 Higher due to proliferation of cells or damage to
reduced clearance from plasma.
 Lower due to reduced synthesis congenital
deficiency.

2.  Each enzyme contain an active site often a water
free cavity, where the substance on which
enzyme acts. substrate interacts with a particular
charged amino acid residues.
 An allosteric site cavity other than active site
whereby regulator molecules bind.
 Co-factor a non-protein part of enzyme
 Inorganic cofactor magnesium, chloride ions
 Co-enzyme organic cofactor such NAD

3. Isoenzymes
 An enzyme may exist in different forms
 Different from each other based on certain
physical properties such as ectrophoretic
mobility, solubility, resistance or activation.
 Isoenzymes are multiple forms of enzymes
arising from genetically determined differences in
primary structure.
 Multiple forms can differ in various ways
 They can be genetically independent protiens
such as cystolic and mitochondrial form of AST.
 The enzyme can be hybrid of one or more
polypeptide chain non-covalently bound e.g LDH,
CK.

4. Utility of Enzyme Levels
 Assessment of cell damage and proliferation
Plasma enzyme levels depend on the rate of
release from damaged cells and if there is no cell
damage then the levels indicate the rate of cell
proliferation or the enzyme synthesis.
 Localisation of damage
Mesurement of plasma activity of an enzyme
known to be in high concentration with in cells of
a paricular tissue may indicate an abnormality of
those cells.

5. Non -specific causes of raised
plasma enzyme activity
 It is important to exclude the presence of non-
specific causes before attributing a change in a
plasma enzyme activity to a specific disease.
 Artefactual increase may occur in haemolysed
samples
 Slight rise in Aspartate transaminase after
moderate exercise
 Rise in CK after a large intramuscular injection.

6. Measurement of Enzyme
Activity
 There are two general methods used to measure
the extent of enzyme activity
 Fixed time reactions
 Continuous monitoring or kinetic assays
 Fixed time reactions
 The reactants are combined, the reaction
proceeds for a designated time, then the reaction
is stopped usually by inactivating the enzyme with
a weak acid and measurement is made. the
reaction is considered linear.
 e.g. creatinine ,blood urea estimation

7. Continuous Monitoring OR kinetic
assay
 The progress of reaction is monitored
continuously through multiple measurements
usually of absorbance change are made during
reaction at specific time interval every 30s60s or
continuously recording spectrophotometer.
 The progress of conversion of substrate into
products in the presence of enzyme is monitored
through measurement of decreasing
concentration of substrate or the increasing
concentration of products.
 Measurement of product formation is
preferable.
 E.g CPK, LDH, SGOT (AST), SGPT (ALT)

8. Diagnostic Enzymology
 Enzyme estimation are useful in the diagnosis
and monitoring of
 Myocardial infarction (CK ,LDH and its
isoenymes ,AST
 Liver disease (ALP, gamma glutamyl transferase
 Bone disease: ricketts, osteomalacia ALP
 Malignancies e.g Prostatic carcinoma (tartrate-
labile acid phosphatase ACP), liver metastasis,
bile duct carcinoma,
 Acute pancreatitis (Amylase)
 Muscle disorders (CK)

9. Types of Plasma Enzymes
Functional plasma
enzymes
Non-functional
Concentration in plasma Present in high conc. in
plasma as compared to
tissues
Normally, present in
plasma in a very low
conc.
function Have known function Not known function
The substrates There substrates are
always present in blood
There substrates are
absent from blood
Site of synthesis liver Different organs e.g
liver,heart, brain and
skeletal muscles ,
prostate
Effect of disease Decrease in liver disease Different enzymes
increase in different
organ diseases
Examples Clotting factors .g
prothrombin, lipoprotien
lipase and
ALT,AST CK,LDH
ALP,ACP and amylase.

10. Sources of non-functional
enzymes
 Increase in the rate of enzyme synthesis e.g
bilirubin increase the rate of synthesis of ALP in
obstructive liver disease.
 Increase permeability of cell membrane as in
tissue hypoxia
 Cell damage with release of its contents into
blood e.g MI, Viral hepatitis

11. Medical importance of non-
functional Plasma Enzymes
 Measurement of plasma enzymes is important
for:
 Diagnosis of disease
 Prognosis of disease ; we can follow up the
effect of treatment by measuring plasma enzymes
before and after treatment.

12. Classification of Enzymes
 Enzymes are classified into six groups according
to type of reaction they catalyze
 1.Oxidoreductases
 2. Transferases
 3.Hydrolases
 4.Lyases
 5.Isomerases
 6.Ligases

13. CK (Creatine kinase) OR creatine
phosphokinase
 Molecular weight =82,000
 Associated with ATP regeneration in contractile
and transport systems
 It involves in the storage of high energy creatine
phosphate in muscle cells.
Tissue source: skeletal muscle, heart muscle,
brain tissue smaller amount in bladder, lung
prostate, kidney.