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Basic enzyme

Basic enzyme. Aulanni’am Biochemistry Laboratory Brawijaya University. What are enzymes ?. Enzymes are proteins They have at least one active site Active site is lined with residues and sometimes contains a co-factor Active site residues have several important properties:

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Basic enzyme

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  1. Basic enzyme Aulanni’am Biochemistry Laboratory Brawijaya University Aulani " Biokimia Enzim " Presentasi 1

  2. What are enzymes ? • Enzymes are proteins • They have at least one active site • Active site is lined with residues and sometimes contains a co-factor • Active site residues have several important properties: • Charge [partial, dipoles, helix dipole] • pKa • Hydrophobicity • Flexibility • Reactivity (Cysteines) Aulani " Biokimia Enzim " Presentasi 1

  3. What are chemical reactions? • In a chemical reactions a compound “A” is changed into a compound “B”. • In context of biochemistry, chemical reactions are “organic chemistry reactions”. • In organic chemistry reactionsbonds are broken and/or formed (generalization) • Bonds are “paired electrons” between two nuclei (C-C, C=C, C-O,C=O, C-H, O-H, N-H etc.) • Thus reactions involve “rearranging” electrons • In context of biochemistry, a frequent player in chemical reactions is H2O (hydronium H3O+ and hydroxide OH-) Aulani " Biokimia Enzim " Presentasi 1

  4. Enzyme catalysis Enzyme catalysis is characterized by two features • Substrate specificity • Rate acceleration Aulani " Biokimia Enzim " Presentasi 1

  5. Enzyme substrate specificity Unlike “chemical catalysts” enzyme only catalyze reactions for a “relatively” narrow substrate spectrum. For example: substrate spectrum of restriction enzymes, and protein kinases. Two main theories for substrate specificity • Lock-and-Key hypothesis (Fisher, 1894) • Induced-fit hypothesis (Koshland, 1958) Aulani " Biokimia Enzim " Presentasi 1

  6. Substrate Transition state Product If enzyme just binds substrate then there will be no further reaction X Enzyme not only recognizes substrate, but also induces the formation of transition state Aulani " Biokimia Enzim " Presentasi 1

  7. B A The Nature of Enzyme Catalysis ●Enzyme provides a catalytic surface ●This surface stabilizes transition state ●Transformed transition state to product B A Catalytic surface Aulani " Biokimia Enzim " Presentasi 1

  8. Lock-and-Key vs. Induced-Fit • Lock-and-Key does not always explain substrate spectrum (e.g. analogs smaller than substrate don’t bind while analogs larger than substrate do bind) • Induced-fit implies the concepts: • conformational change • catalytically competent conformation (low catalytic form and high catalytic form) Aulani " Biokimia Enzim " Presentasi 1

  9. S‡ S‡c Free Energy (delta G) ES‡ S ES EP P Reaction Coordinate Catalyzed vs. un-catalyzed reactions Aulani " Biokimia Enzim " Presentasi 1

  10. + + N H N H O = O O C = = O C C H C H = N H H C H H C H C N H N H H C H O O O N H O H H H H H H H H - - O O O O C C Induced to transition state Specific Acid-base Catalysis Acid catalysis +d Both -d Base catalysis Slow Fast Fast Very Fast Aulani " Biokimia Enzim " Presentasi 1

  11. S‡ Free Energy (delta G) ES‡ S ES EP P Reaction Coordinate Rate Acceleration • Catalyzes of a reaction results in rate enhancementnot alteration of the equilibrium • Catalysis involves reduction of activation energy • This can be most readily done by lowering the Free Energy of the transition state • Additionally the Free Energy of the ground state can be raised (not a general strategy) Aulani " Biokimia Enzim " Presentasi 1

  12. S‡ Free Energy (delta G) ES‡ S ES EP P Reaction Coordinate Transition state Stabilization by Enzyme How does an Enzyme reduce the Activation Energy ?? • Enzyme stabilizes the transition state, i.e. makes the “strained” conformation more bearable. Note: • An enzyme can only reduce the activation energy if it binds better to the transition state than to the substrate [otherwise, the DDG between ES and ES‡ is the same as between S and S‡] Aulani " Biokimia Enzim " Presentasi 1

  13. S‡ Free Energy (delta G) ES‡ S ES EP P Reaction Coordinate Transition state Stabilization by Enzyme Implications of preferential stabilization of the transition state. • Compounds that closely mimic the transition state bind much better to an enzyme than the original substrate. • Transition state analogs are potent inhibitors (pico molar affinities) • Applications: • Inhibitor/drug development based on transition state model • Development of catalytic antibodies [rate acceleration up to 105] Aulani " Biokimia Enzim " Presentasi 1

  14. Reaction direction Enzyme Stabilizes Transition State Energy change ST Energy decreases (under catalysis) Energy required (no catalysis) EST S ES P EP T = Transition state What’s the difference? Aulani " Biokimia Enzim " Presentasi 1

  15. 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 CoE (2) (1) (3) Reactive groups (4) (4) Coenzyme helps - (3) Aulani " Biokimia Enzim " Presentasi 1

  16. 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 Aulani " Biokimia Enzim " Presentasi 1

  17. Enzyme mediated catalysis • Strategies for transition state stabilization and/or ground state destabilization: • Proximity • Strain or distortion • Orbital steering • However, additionally the enzyme can be an “active” participant in reaction • Acid/base catalysis • Nucleophilic/electrophilic catalysis • Covalent catalysis Aulani " Biokimia Enzim " Presentasi 1

  18. Rate Acceleration: Proximity • For un-catalyzed reactions involving two substrates the rate can be increased by increasing the number of collisions (higher temperature) • Enzymes capture each substrate (sometimes in a ordered manner) and appropriately orient them with respect to each other, thus obviating the need for higher temperature • The capture of substrates by the enzyme has an Entropic cost; this cost must be compensated by favourable interactions between enzyme and substrates • The effect of confining the substrates in the active site of the enzyme is similar to raising the concentration of the substrates. Hence, the proximity effect is also referred to as increasing the effective concentration Aulani " Biokimia Enzim " Presentasi 1

  19. Active Site Avoids the Influence of Water + - Preventing the influence of water sustains the formation of stable ionicbonds Aulani " Biokimia Enzim " Presentasi 1

  20. E S Essential of Enzyme Kinetics Steady State Theory E E + + P S In steady state, the production and consumption of the transition state proceed at the same rate. So the concentration of transition state keeps a constant. Aulani " Biokimia Enzim " Presentasi 1

  21. Concentration Reaction Time Constant ES Concentration at Steady State S P ES E Aulani " Biokimia Enzim " Presentasi 1

  22. Weak electrophile Poor nucleophile The “Active” Enzyme Examine the hydrolysis of an ester: Expected transition state Aulani " Biokimia Enzim " Presentasi 1

  23. The “Active” Enzyme Base catalyzed hydrolysis of an ester: Catalysis is accelerated by altering the poor nucleophile H2O into a strong nucleophile OH- Aulani " Biokimia Enzim " Presentasi 1

  24. The “Active” Enzyme Acid catalyzed hydrolysis of an ester: Catalysis is accelerated by altering the weak electrophile C into a strong nucleophile C+ Aulani " Biokimia Enzim " Presentasi 1

  25. The “Active” Enzyme • In standard organic chemistry for ester hydrolysis one has to choose between base or acid catalysis • In enzyme catalysis the reaction is “carried out on a solid support” • As a consequence one can incorporate both acid and base catalysis: Aulani " Biokimia Enzim " Presentasi 1

  26. The “Active” Enzyme • Enzyme catalyzed hydrolysis of an ester: • Active site incorporates both: • a base [-B:] • an acid [-B+-H] Aulani " Biokimia Enzim " Presentasi 1

  27. Catalysis of Phosphorylation • Phosphorylation a very frequent reaction (e.g. signal transduction) • Phosphoryl donating group is generally a nucleotide, e.g. ATP, GTP • Transfer of phosphoryl group to: • Water : hydrolysis [ATPase, GTPase] • Anything else: phosphorylation [Kinase] Aulani " Biokimia Enzim " Presentasi 1

  28. Mechanisms of Enzyme Catalyzed Phosphorylation • Several mechanism are observed in Nature • Reactions with covalent enzyme intermediates • Direct inline transfer • Perhaps metal assisted mechanisms • Present two examples: • Aminoglycoside kinases (Cousin of Protein kinases) • G-proteins Aulani " Biokimia Enzim " Presentasi 1

  29. 1 vo vo - 1 Km 1 Vmax 1/S S An Example for Enzyme Kinetics (Invertase) 1) Use predefined amount of Enzyme→ E 2)Add substrate in various concentrations → S (x 3)Measure Product in fixed Time (P/t) →vo (y 4) (x, y) plot get hyperbolic curve, estimate→ Vmax 5)When y = 1/2 Vmaxcalculate x ([S]) →Km Vmax 1/2 Km Double reciprocal Direct plot Aulani " Biokimia Enzim " Presentasi 1

  30. A Real Example for Enzyme Kinetics Substrate Product Velocity Double reciprocal 2.0 1.0 0 1.0 0.5 0 v 1/v 1.0 -3.8 -4 -2 0 2 4 0 1 2 [S] 1/[S] Data v (mmole/min) [S] Absorbance 1/S 1/v no 0.25 0.50 1.0 2.0 0.21 0.36 0.40 0.46 0.42 0.72 0.80 0.92 4 2 1 0.5 2.08 1.56 1.35 1.16 1 2 3 4 → → → → (1) The product was measured by spectroscopy at 600 nm for 0.05 per mmole (2) Reaction time was 10 min Direct plot Double reciprocal Aulani " Biokimia Enzim " Presentasi 1

  31. Enzyme Inhibition (Mechanism) E + S→ES→E + P + I ↓ EI E + S→ES→E + P + + II ↓ ↓ EI+S→EIS E + S→ES→E + P + I ↓ EIS ← ← ← ↑ ↑ ↑ ↑ Competitive Uncompetitive Non-competitive E Substrate E X Cartoon Guide Compete for active site Inhibitor Different site Equation and Description [I] binds to free [E] only, and competes with [S]; increasing [S] overcomes Inhibition by [I]. [I] binds to [ES] complex only, increasing [S] favors the inhibition by [I]. [I] binds to free [E] or [ES] complex; Increasing [S] can not overcome [I] inhibition. Aulani " Biokimia Enzim " Presentasi 1

  32. Enzyme Inhibition (Plots) Uncompetitive Competitive Non-competitive Vmax Vmax vo Vmax’ Vmax’ I Direct Plots Km [S], mM Km’ Km [S], mM 1/vo 1/vo 1/vo I I Double Reciprocal Two parallel lines Intersect at X axis Intersect at Y axis 1/Vmax 1/Vmax 1/Vmax 1/Km 1/[S] 1/Km 1/[S] 1/Km 1/[S] Vmax vo I I Km Km’ [S], mM = Km’ Vmax unchanged Km increased Vmax decreased Km unchanged Both Vmax & Km decreased I Aulani " Biokimia Enzim " Presentasi 1

  33. H O C–O–H O C–O- C = = N H–N H–O–CH2 C C H CH2 H C Ser 195 N–H N -O–CH2 C C Asp 102 H CH2 His 57 Ser 195 Asp 102 His 57 Active Ser Aulani " Biokimia Enzim " Presentasi 1

  34. 5 6 7 8 9 10 11 pH pH Influences Chymotrypsin Activity Relative Activity Aulani " Biokimia Enzim " Presentasi 1

  35. 10 9 8 7 6 5 4 3 Buffer pH pH Influences Net Charge of Protein Isoelectric point, pI + 0 + - Net Charge of a Protein Aulani " Biokimia Enzim " Presentasi 1

  36. Imidazole on Histidine Is Affected by pH H H O C–O- = H–O–CH2 C C N–H N H–N H–N H+ C C C C H H H + Ser 195 C N–H H–N C C-H Asp 102 CH2 His 57 pH 6 pH 7 + Inactive Aulani " Biokimia Enzim " Presentasi 1

  37. Chymotrypsin Produces New Ile16 N-Terminal Relative activity L13 I16 Y146 pH 5 6 7 8 9 10 11 Ile 16 Ile 16 NH2– Asp 194 –CH2COO- pH 9 pH 10 +NH3– New NH2-terminus pKa Aulani " Biokimia Enzim " Presentasi 1

  38. New Ile16 N-Terminal Stabilizes Asp194 Catalytic Triad Gly 193 His 57 Ser 195 Asp 194 Asp 102 +NH3 Ile 16 Aulani " Biokimia Enzim " Presentasi 1

  39. Chymotrypsin Ser195 Inhibited by DIFP O (CH3)2CH–O–P–O–CH(CH3)2 F = O (CH3)2CH–O–P–O–CH(CH3)2 = O-…H CH2 Ser 195 O CH2 Ser 195 Diisopropyl-fluorophosphate (DIFP) X Aulani " Biokimia Enzim " Presentasi 1

  40. Addition of Substrate Blocks DIFP Inhibition 100 50 0 Percent Inhibition of activity (%) No substrate + DIFP X Add substrate + DIFP & substrate S Reaction time Aulani " Biokimia Enzim " Presentasi 1

  41. Chymotrypsin Also Catalyzes Acetate O CH3–C–O– –NO2 O -C N- H O CH3–C–OH O -C O- HO– –NO2 Hartley & Kilby Nitrophenol acetate + H2O Chymotrypsin Peptide bond Acetate Nitrophenol Ester bond No acetate was detected at early stage Aulani " Biokimia Enzim " Presentasi 1

  42. Two-Stage Catalysis of Chymotrypsin O CH3–C–O– –NO2 O C O- C O-H C O CH3–C HO– –NO2 Nitrophenol Time (sec) Acylation Nitrophenol acetate Kinetics of reaction Deacylation (slow step) CH3COOH + H2O Two-phase reaction Aulani " Biokimia Enzim " Presentasi 1

  43. Extra Negative Charge Was Neutralized -C-C-N-C-C-N-C-C-N- H H O- -C N- HOH O- -C N- HOH O -C N- H O -C-OH NH2- E + S Aulani " Biokimia Enzim " Presentasi 1

  44. Active Site Stabilizes Transition State Gly 193 Ser 195 Asp 194 Met 192 His 57 Cys 191 Asp 102 Thr 219 Cys 220 Ser 214 Trp 215 Gly 216 Ser 218 Ser 217 Catalytic Triad Active Site Specificity Site Aulani " Biokimia Enzim " Presentasi 1

  45. Regulation of Enzyme Activity Inhibitor Proteolysis or inhibitor Feedback regulation Phosophorylation P Signal transduction Regulatory subunit (-) proteolysis P R R (+) regulator effector phosphorylation or + cAMP or calmodulin Aulani " Biokimia Enzim " Presentasi 1

  46. Classification of Proteases 2+ Zn Carboxy- peptidase A Non- polar EDTA EGTA H196 E72 H69 S195-O- DFP TLCK TPCK Aromatic Basic Chymotrypsin Trypsin H57 D102 C25-S- PCMB Leupeptin Non- specific Papain H195 D215 Pepsin Renin Non- specific Pepstatin H2O D32 Family Example Mechanism Specificity Inhibitor Metal Protease Serine Protease Cysteine Protease Aspartyl Protease Aulani " Biokimia Enzim " Presentasi 1

  47. 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 – Ser 195 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 – – – – – – – – His 57 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 – – – – – – – Asp 102 Aulani " Biokimia Enzim " Presentasi 1

  48. Sigmoidal Curve Effect vo ATP CTP vo Noncooperative (Hyperbolic) Positive effector (ATP) brings sigmoidal curve back to hyperbolic Negative effector (CTP) keeps Sigmoidal curve Cooperative (Sigmoidal) Exaggeration of sigmoidal curve yields a drastic zigzag line that shows the On/Off point clearly Consequently, Allosteric enzyme can sense the concentration of the environment and adjust its activity Off On Aulani " Biokimia Enzim " Presentasi 1 [Substrate]

  49. Mechanism and Example of Allosteric Effect Kinetics Models Cooperation vo (+) [S] (+) vo (+) [S] vo (-) (-) [S] Allosteric site R = Relax (active) Homotropic (+) Concerted Allosteric site A Heterotropic (+) Sequential X I Heterotropic (-) Concerted T = Tense (inactive) X X Aulani " Biokimia Enzim " Presentasi 1

  50. A A P P P P A A A A P P P P P P A A Activity Regulation of Glycogen Phosphorylase Covalent modification GP kinase T P T P GP phosphatase 1 Non-covalent ATP Glc-6-P Glucose Caffeine spontaneously A Glucose Caffeine AMP A R R A Aulani " Biokimia Enzim " Presentasi 1

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