1. ENZYMES
-Keshava Pavan K

2. • Prosthetic group
– tightly bound coenzymes
– FAD, FMN, Biotin
• Cosubstrates
– loosely bound coenzymes
– NAD+, NADP+

3. • Coenzymes transferring H :
– FAD, FMN, NAD, NADP- water soluble vitamins
– Tetrahydrobiopterin
– Lipoic acid
– Coenzyme Q

4. • Coenzymes transferring groups other than H
– Biotin (CO2)
– Pyridoxal phosphate (amino)
– CoA (acyl)
– Tetrahydro folic acid (1 C groups)
– Methyl cobalamine (CH3)
– ATP (PO4)
– S Adenosyl methionine (CH3)
– UDP (glucose/galactose)
• Last three do not belong to Vitamin B complex

5. • Activators are metal ions
– tightly bound- metalloenzymes
– loosely bound- metal activated enzymes
• Stabilise proper conformation
• Cu2+ for tyrosinase, Mg2+ for kinases
• Fe, Cu in oxidation-reduction reactions
• Pyruvate dehydrogenase complex requires 5
activators: Mg2+, NAD+, FAD+, TPP, CoA
• Xanthine oxidase requires FAD, Mo, Fe

6. • Multifunctional enzymes: same enzyme
molecule having different functions for
different parts of that enzyme molecule
– fatty acid synthase complex
• Multienzyme complex: many enzymes
clustered together having common function

7. • Active site of lysozyme:
Glu Asp Trp Trp Asp
35 52 62 63 101
Catalytic site substrate binding site
• Hexokinase- 0.02 mmol L-1 : always active
• Glucokinase- 10 mmol L-1 : only when glucose
concentration is high, only in liver

8. CLINICAL APPLICATIONS OF COMPETITIVE INHIBITION
• Sulphonamides(NH2-C6H5-SO2-NH2) resemble
PABA (NH2-C6H5-COOH), hence inhibits it
• Anticancer drugs like methotrexate,
aminopterin inhibit dihydrofolate reductase
• Alcohol dehydrogenase catalyses conversion
of methyl alcohol to formaldehyde. Ethyl
alcohol inhibits it. Therefore ethyl alcohol is
used during methyl alcohol poisoning.

9. • Allopurinol is used to treat gout as it inhibits
xanthine oxidase that converts hypoxanthine
to uric acid
• Dicoumarol is an anticoagulantas it inhibits
Vit K needed for coagulation
• Lovastatin inhibits HMG-CoA reductase, hence
used to reduce cholesterol level in blood

10. NONCOMPETITIVE INHIBITORS
• Cyanide inhibits cytochrome oxidase
• Iodoacetate inhibits enzynes having –SH
(sulfhydryl) group at active site like
glyceraldehyde-3-phosphate dehydrogenase
• Fluoride inhibits enolase (binding with Mg 2+
or Mn2+)
• Di-isopropyl fluorophosphate (DFP) inhibits
enzymes having serine at active site like
chymotrypsin, acetylcholine esterase

11. SUICIDE INHIBITION
• Irreversible
• Mechanism based inhibition
• Allopurinol becomes alloxanthine which is a
more potent inhibitor
• 5-fluoro uracil becomes fluoro deoxy uridylate
which binds to the enzyme thymidylate
synthetase and inhibits it

12. ALLOSTERIC MODULATION
• Regulation of enzyme activity in the body
• Positive & negative modulators
• Not a substrate analog
• Reversible
• Allosteric site other than active site
• Brings about conformational change
• Mostly oligomeric enzymes
– Aspartate transcarbamoylase (6 polypeptide chains)
– Pyruvate kinase (4 polypeptide chains)

13. • Have sigmoidal curve
ENZYME INHIBITOR ACTIVATOR
ALA synthase heme
phosphofructokinase ATP, citrate AMP
Aspartate transcarbamoylase CTP ATP

14. UNCOMPETITIVE INHIBITION
• Cannot bind with free enzyme, only to E-S
complex
inhibitor
• 1/V
1/S
• Eg,. Inhibition of placental alkaline
phosphatase (Regan isoenzyme) by phenyl
alanine

15. REGULATION OF ENZYME ACTIVITY
• Induction (depression) and repression
– Some are constitutive enzymes like hexokinase
– Others are inducible like glucokinase
• Allosteric modulation
• Covalent modulation
– Zymogen activation, phosphorylation and
dephosphorylation
• Compartmentalisation of pathways
• Degradation
• isoenzymes

16. DIAGNOSTIC ENZYMES
• Aspartate transaminase
– Normal 2 – 40 IU/L
– Increases during MI & liver diseases
• Alanine transaminase
– Normal 0 – 45 IU/L
– Very high during liver diseases
– Moderately high during MI

17. • Alkaline phosphatase
– Hydrolysis of phosphate esters
– Optimum pH 9.5-10
– 25-100 IU/L
– Increases in liver & bone diseases
– Very high in cholestatic/obstructive jaundice
• Acid phosphatase (prostatic fraction)
– Optimum pH 4.5
– Increases in bone diseases & prostate cancers
– Prostatic specific antigen-protease
• 5mg/L
• 4 to 10mg/L benign; >10 mg/L cancerous

18. • 5’ nucleotidase
– Normal 2-10 IU/L
– High in liver diseases, very high in cholestasis
• γ- glutamyl transpeptidase
– Normal 10-30 IU/L
– High in liver diseases, very high in alcoholics
• Cholinesterase
– Normal 2-121 IU/L
– Inhibited by organophosphorous pesticides
• Pseudocholinesterase
– Decreases in liver diseases

19. • Amylase
– Normal 50-120 IU/L
– Very high in acute pancreatitis
– Moderately high in chronic pancreatitis & mumps
• Lipase
– Normal 0.2-1.5 IU/L
– Diagnostics same as above; also high in cholestasis

20. THERAPEUTIC ENZYMES
• Streptokinase
– To dissolve intravascular clots
• Asparginase
– To treat leukemia
• Pancreatin
– Lipase, trypsin
– To treat pancreatic insufficiency
• Collagenase
– To remove scar tissue

21. ANALYTICAL ENZYMES
• Glucose oxidase, peroxidase
– Estimation of glucose
• Cholesterol oxidase
– Estimation of cholesterol
• Horse- raddish peroxidase- ELISA
• Taq polymerase- PCR
• Restriction endonuclease- rDNA technology &
DNA fingerprinting

22. RIBOZYMES
• snRNA/small nuclear RNA
– Splicing
• Peptidyl transferase
– translation