Enzypes for MBBS students of first year.I am very thankful to all the colleagues

2. ENZYMES © 2007 Paul Billiet ODWS BERZELIUS 1835 Starch. Hydrolysis. KUHNE 1878 Enzyme mean yeast. EDWARD BUCHNER Sucrose to Ethanol

3. ENZYMES © 2007 Paul Billiet ODWS EDWARD BUCHNER N.P.1907 ARTHUR HARDEN N.P. 1929 JAMES SUMNER N.P. 1946 JOHAN NORTHROP N.P. 1946 WILHELM OSTWALD N.P. 1909 WARBURG N.P. 1970

7. ENZYMES © 2007 Paul Billiet ODWS EDWARD BUCHNER N.P.1907 ARTHUR HARDEN N.P. 1929 JAMES SUMNER N.P. 1946 JOHAN NORTHROP N.P. 1946 WILHELM OSTWALD N.P. 1909 WARBURG N.P. 1970

15. TABULAR FORM SHOWING CO.E

24. TISSUES BRAIN,HEART,LIVER,KIDNEY,MUSCLE MUSCLE -> ← HEART -> LIVER ← STOMACH BRAIN ← KIDNEY ← INTESTINE

31. What is a Ribozyme? 1) Enzyme 2) Ribonucleic Acid NOT PROTEIN 1989 Nobel Prize In Chemistry Sid Altman Tom Cech

35. Structure : As with proteins, we consider... Primary: GGCCGAACUGGUA Secondary: Tertiary:

36. Secondary Structure Watson-Crick Base Pairing Helix Formation B-DNA Small pore along helical axis “ Rungs” stack obliquely to axis A-DNA RNA RNA usually assumes A-form helices…

38. Ribozyme vs. tRNA Phe folding

39. Tertiary Structure

40. The Future of Ribozymes In Vitro Molecular Evolution of RNA High Throughput Screening Ribozyme-Based Therapies +

41. In Clinical Trial... HIV Gene Therapy... Bone Marrow Sample Treat Stem Cells with Retroviral Vector Re-Implant Treated Cells Encodes Gene for anti-HIV Ribozyme

46. Enzyme Stabilizes Transition State S P ES ES T EP S T Energy change Energy required (no catalysis) Energy decreases (under catalysis) Sub.(S) Prod. (P)Enz(E) T = Transition state V=rate of change of S to P/mt. Adapted from Alberts et al (2002) Molecular Biology of the Cell (4e) p.166 Reaction direction

48. Control Points of Gene Regulation Prokaryotics Post-translational control Eukaryotics Juang RH (2004) BCbasics DNA ribosome mRNA proteins proteins cap 5’ 3’ tail mature mRNA DNA 5’ 3’ process mRNA Translation Activity Proteolysis Transcription RNA Processing RNA Transport RNA Degradation

51. Active Site Avoids the Influence of Water Preventing the influence of water sustains the formation of stable ionic bonds - +

52. Active Site Is a Deep Buried Pocket Why energy required to reach transition state is lower in the active site? It is a magic pocket (1) Stabilizes transition (2) Expels water (3) Reactive groups (4) Coenzyme helps (2) (3) (4) (1) CoE + - Juang RH (2004) BCbasics

55. ACTIVE SITE

74. The effect of temperature Temperature / °C Enzyme activity 0 10 20 30 40 50 Q10 Denaturation

77. The effect of pH Optimum pH values © 2007 Paul Billiet ODWS Enzyme activity Trypsin Pepsin pH 1 3 5 7 9 11

84. Sigmoidal Curve Effect Sigmoidal curve Exaggeration of sigmoidal curve yields a drastic zigzag line that shows the On/Off point clearly Positive effector (ATP) brings sigmoidal curve back to hyperbolic Negative effector (CTP) keeps Consequently, Allosteric enzyme can sense the concentration of the environment and adjust its activity Noncooperative (Hyperbolic) Cooperative (Sigmoidal) v o [Substrate] Off On Juang RH (2004) BCbasics CTP ATP v o

92. Regulation of Enzyme Activity P R R + proteolysis phosphorylation cAMP or calmodulin or regulator effector (+) Juang RH (2004) BCbasics Regulatory subunit or inhibitor P (-) Inhibitor Proteolysis Phosophorylation Signal transduction Feedback regulation

103. CCC Allosteric Enzyme ATCase + Active relaxed form Inactive tense form ATCase R R R R R R CCC Catalytic subunits Catalytic subunits Regulatory subunits Aspartate Carbamoyl phosphate Carbamoyl aspartate Juang RH (2004) BCbasics Quaternary structure COO - CH 2 HN-C-COO - H H - - - - O H 2 N-C-O-PO 3 2- = O H 2 N-C- = COO - CH 2 N-C-COO - H H - - - - ATP CTP Nucleic acid metabolism Feedback inhibition CTP CTP CTP CTP CTP CTP

105. The Reception and Transduction of Signals -GDP +GTP Adenylate cyclase   Glycogen Synthase active Insulin kinase Glucagon A The third group: Ion-channel-linked Receptor Gilman, Rodbell (1994) Glycogen breakdown Juang RH (2007) BCbasics SH2 domain  G protein   GDP   + Signal  GDP  GTP  GTP + Signal Activation P Protein Phosphatase Glycogen Synthase P P P P P G-protein-linked Receptor Enzyme-linked Receptor Glycogen

106. Sigmoidal Curve Effect Sigmoidal curve Exaggeration of sigmoidal curve yields a drastic zigzag line that shows the On/Off point clearly Positive effector (ATP) brings sigmoidal curve back to hyperbolic Negative effector (CTP) keeps Consequently, Allosteric enzyme can sense the concentration of the environment and adjust its activity Noncooperative (Hyperbolic) Cooperative (Sigmoidal) v o [Substrate] Off On Juang RH (2004) BCbasics CTP ATP v o

110. cAMP Controls Activity of Protein Kinase A A A A A cAMP Active kinase CREB CREB Nucleus Activation Gene expression ON DNA Alberts et al (2002) Molecular Biology of the Cell (4e) p. 857, 858 R C R C R R A A A A C C Regulatory subunits Catalytic subunits C P

114. INHIBITORS

125. The allosteric site the enzyme “on-off” switch Active site Allosteric site empty Substrate fits into the active site The inhibitor molecule is absent Conformational change Inhibitor fits into allosteric site Substrate cannot fit into the active site Inhibitor molecule is present © 2008 Paul Billiet ODWS E E

132. Enzyme Inhibition (Mechanism) Competitive Non-competitive Uncompetitive E E Different site Compete for active site Inhibitor Substrate Cartoon Guide Equation and Description [ I ] binds to free [E] only, and competes with [S]; increasing [S] overcomes Inhibition by [ I ]. [ I ] binds to free [E] or [ES] complex; Increasing [S] can not overcome [ I ] inhibition. [ I ] binds to [ES] complex only, increasing [S] favors the inhibition by [ I ]. X Juang RH (2004) BCbasics E + S -> ES -> E + P + I ↓ E I ← ↑ E + S -> ES -> E + P + + I I ↓ ↓ E I + S ->E I S ← ↑ ↑ E + S -> ES -> E + P + I ↓ E I S ← ↑

133. Competitive Inhibition Succinate Glutarate Malonate Oxalate Succinate Dehydrogenase Substrate Competitive Inhibitor Product C-OO - C-H C-H C-OO - C-OO - H-C-H H-C-H C-OO - C-OO - H-C-H H-C-H H-C-H C-OO - C-OO - C-OO - C-OO - H-C-H C-OO -

134. Enzyme Inhibition (Plots) V max K m K m ’ [S], mM v o I I V max unchanged K m increased V max decreased K m unchanged Both V max & K m decreased I = K m ’ Juang RH (2004) BCbasics K m Competitive Non-competitive Uncompetitive Direct Plots Double Reciprocal V max [S], mM v o K m [S], mM V max I K m ’ V max ’ V max ’ 1/[S] 1/K m 1/ v o 1/ V max I Two parallel lines I Intersect at X axis 1/ v o 1/ V max 1/[S] 1/K m 1/[S] 1/K m 1/ V max 1/ v o Intersect at Y axis

138. CLINICAL APPLICATIONS OF COMPETITVE INHIBITORS METHANOL POISONING METHANOL Al.Dehy. ETHANOL ANTIBIOTIC PABA Dihydro pteroate Synthase SULFONAMIDE GOUT HYPOXANTHENE XANTHINE OXIDASE ALLOPURINOL Clinical App. TRUE SUB. ENZYME DRUG

143. Enzyme Inhibitors Are Extensively Used ● Sulfa drug (anti-inflammation) Pseudo substrate competitive inhibitor ● Protease inhibitor Plaques in brain contains protein inhibitor ● HIV protease is critical to life cycle of HIV HIV protease (homodimer): ↑ inhibitor is used to treat AIDS Symmetry Not symmetry -> Human aspartyl protease: (monodimer) Alzheimer's disease domain 1 Asp Asp domain 2 subunit 2 Asp subunit 1 Asp

150. Intracellular Distribution of Diagnostic Enzymes ACP ALP GGT CK ALP AMS LPS AMS LD 1 AST CK LD 5 ALT AST Prostate Biliary Tract Muscle Bone Salivary Glands Pancreas Heart Liver

180. THANKS

181. Competitive Inhibition Succinate Glutarate Malonate Oxalate Succinate Dehydrogenase Substrate Competitive Inhibitor Product C-OO - C-H C-H C-OO - C-OO - H-C-H H-C-H C-OO - C-OO - H-C-H H-C-H H-C-H C-OO - C-OO - C-OO - C-OO - H-C-H C-OO -

182. Enzyme Active Site Is Deeper than Ab Binding Instead, active site on enzyme also recognizes substrate, but actually complementally fits the transition state and stabilized it. Ag binding site on Ab binds to Ag complementally, no further reaction occurs. X

183. cAMP Controls Activity of Protein Kinase A A A A A cAMP Active kinase CREB CREB Nucleus Activation Gene expression ON DNA Alberts et al (2002) Molecular Biology of the Cell (4e) p. 857, 858 R C R C R R A A A A C C Regulatory subunits Catalytic subunits C P

184. HIV protease vs Aspartyl protease Asymmetric monomer ↓ HIV protease (homodimer) HIV Protease inhibitor is used in treating AIDS Symmetric dimer ↑ Aspartyl protease (monomer) Juang RH (2004) BCbasics Asp subunit 2 subunit 1 Asp domain 1 domain 2 Asp Asp

185. Enzyme Inhibitors Are Extensively Used ● Sulfa drug (anti-inflammation) Pseudo substrate competitive inhibitor ● Protease inhibitor Plaques in brain contains protein inhibitor ● HIV protease is critical to life cycle of HIV HIV protease (homodimer): ↑ inhibitor is used to treat AIDS Symmetry Not symmetry -> Human aspartyl protease: (monodimer) Alzheimer's disease domain 1 Asp Asp domain 2 subunit 2 Asp subunit 1 Asp

186. Sulfa Drug Is Competitive Inhibitor Precursor Folic acid Tetrahydro- folic acid Sulfanilamide Sulfa drug (anti-inflammation) Para-aminobenzoic acid (PABA) Bacteria needs PABA for the biosynthesis of folic acid Sulfa drugs has similar structure with PABA, and inhibit bacteria growth. Domagk (1939) -COOH H 2 N- -SONH 2 H 2 N-

187. The Reception and Transduction of Signals -GDP +GTP Adenylate cyclase   Glycogen Synthase active Insulin kinase Glucagon A The third group: Ion-channel-linked Receptor Gilman, Rodbell (1994) Glycogen breakdown Juang RH (2007) BCbasics SH2 domain  G protein   GDP   + Signal  GDP  GTP  GTP + Signal Activation P Protein Phosphatase Glycogen Synthase P P P P P G-protein-linked Receptor Enzyme-linked Receptor Glycogen

188. Signal Transduction Network (Ras vs. P53) Cytosol Cell membrane Effector enzyme Signal protein E2F Transcription factor Target gene mRNA Inhibitor P53 Cell division ON Signal Receptor Nucleus Ribosome Transcription Transcription Apoptosis Cell function are controlled by protein interactions mRNA Regulator protein Juang RH (2007) BCbasics Ras

189. The Reception and Transduction of Signals -GDP +GTP Adenylate cyclase   Glycogen Synthase active Insulin kinase Glucagon A The third group: Ion-channel-linked Receptor Gilman, Rodbell (1994) Glycogen breakdown Juang RH (2007) BCbasics SH2 domain  G protein   GDP   + Signal  GDP  GTP  GTP + Signal Activation P Protein Phosphatase Glycogen Synthase P P P P P G-protein-linked Receptor Enzyme-linked Receptor Glycogen

190. A PKA active inactive Glucagon P P GP kinase GP kinase GP a GP b Glycogen synthase Glycogen synthase P Protein phosphatase-1 Protein phosphatase-1 Protein phosphatase inhibitor-1 Protein phosphatase inhibitor-1 Glycogen P Phosphatase

191. Classification of Proteases Metal Protease Serine Protease Cysteine Protease Aspartyl Protease Family Example Mechanism Specificity Inhibitor Juang RH (2004) BCbasics Carboxy- peptidase A Chymotrypsin Trypsin Papain Pepsin Renin H57 D102 S195-O - C25-S - H195 D215 D32 H 2 O Non- specific Non- specific Aromatic Basic Non- polar EDTA EGTA DFP TLCK TPCK PCMB Leupeptin Pepstatin E72 H69 Zn 2+ H196

192. Modification of Subtilisin and Its Activity Change No enzyme 1 Asn 155 -> Leu ● ● ● 10,000,000 ( Asn 155 stabilizes transition state ) His & Asp -> Ala ● ○ ○ 37,000 Ser , His & Asp -> Ala ○ ○ ○ 4,000 Subtilisin ● ● ● 10,000,000,000 Active Site Relative Modification Triad: Ser His Asp activity Ser -> Ala ○ ● ● 5,000 Asp -> Ala ● ● ○ 330,000

193. Serine Protease and AchE Chymotrypsin – Gly – Asp – Ser – Gly – Gly – Pro – Leu – Trypsin – Gly – Asp – Ser – Gly – Gly – Pro – Val – Elastase – Gly – Asp – Ser – Gly – Gly – Pro – Leu – Thrombin – Gly – Asp – Ser – Gly – Gly – Pro – Phe – Plasmin – Gly – Asp – Ser – Gly – Gly – Pro – Leu – Acetylcholinesterase – Gly – Glu – Ser – Ala – Gly – Gly – Ala – Chymotrypsin – Val – Thr – Ala – Ala – His – Cys – Gly – Trypsin – Val – Ser – Ala – Gly – His – Cys – Tyr – Elastase – Leu – Thr – Ala – Ala – His – Cys – Ile – Thrombin – Leu – Thr – Ala – Ala – His – Cys – Leu – Plasmin – Leu – Thr – Ala – Ala – His – Cys – Leu – Acetylcholinesterase – – – – – – – – – – – – – His – – – – – – – – Ser 195 Chymotrypsin – Thr – Ile – Asn – Asn – Asp – Ile – Thr – Trypsin – Tyr – Leu – Asn – Asn – Asp – Ile – Met – Elastase – Ser – Lys – Gly – Asn – Asp – Ile – Ala – Thrombin – Asn – Leu – Asp – Arg – Asp – Ile – Ala – Plasmin – Phe – Thr – Arg – Lys – Asp – Ile – Ala – Acetylcholinesterase – – – – – – – – – – – – – – Asp – – – – – – – His 57 Asp 102 Adapted from Dressler & Potter (1991) Discovering Enzymes, p.244

194. H AchE Has Similar Catalytic Mechanism H - O - H H 2 O Adapted from Dressler & Potter (1991) Discovering Enzymes, p.243 ↓ Deacylation Acylation↑ AchE O - C H O CH 3 CH 3 – C – O–CH 2 –CH 2 – N –CH 3 CH 3 + AchE O C H O CH 3 – C CH 3 H O–CH 2 –CH 2 – N –CH 3 CH 3 + AchE O - C H H O CH 3 – C – OH AchE O - C H O CH 3 – C CH 3 O–CH 2 –CH 2 – N –CH 3 H CH 3 +

195. Different Enzymes Might Adopt Same Mechanism Hi, Everybody! ← Useful ↙ Amusing Juang RH (2004) BCbasics O - C Sesame Triad

196. Convergent and Divergent Trypsin Chymotrypsin Elastase Thrombin Plasmin Acetylcholin esterase Thyroglobulin Ester bond Peptide bond hydrolyze acetylcholine Serine Protease Juang RH (2004) BCbasics Divergent evolution Asp-- His-- Ser Asp--His--Ser Convergent evolution C N C C H O C C C O O Evolution Molecular

197. Activity Regulation of Glycogen Phosphorylase Covalent modification P P GP kinase GP phosphatase 1 Non-covalent A A A AMP ATP Glc-6-P Glucose Caffeine Glucose Caffeine spontaneously R T R T Garrett & Grisham (1999) Biochemistry (2e) p.679 P A P A P P A A P A P A P P P A P A

198. CCC Allosteric Enzyme ATCase + Active relaxed form Inactive tense form ATCase R R R R R R CCC Catalytic subunits Catalytic subunits Regulatory subunits Aspartate Carbamoyl phosphate Carbamoyl aspartate Juang RH (2004) BCbasics Quaternary structure COO - CH 2 HN-C-COO - H H - - - - O H 2 N-C-O-PO 3 2- = O H 2 N-C- = COO - CH 2 N-C-COO - H H - - - - ATP CTP Nucleic acid metabolism Feedback inhibition CTP CTP CTP CTP CTP CTP

199. Regulation of Enzyme Activity P R R + proteolysis phosphorylation cAMP or calmodulin or regulator effector (+) Juang RH (2004) BCbasics Regulatory subunit or inhibitor P (-) Inhibitor Proteolysis Phosophorylation Signal transduction Feedback regulation