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

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Presentation on theme: "Aulani " Biokimia Enzim " Presentasi 1 1 Basic enzyme Aulanni’am Biochemistry Laboratory Brawijaya University."— Presentation transcript:

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

2 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 )

3 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 reactions bonds 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 H 2 O (hydronium H 3 O + and hydroxide OH - )

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

5 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)

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

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

8 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)

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

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

11 Aulani " Biokimia Enzim " Presentasi 1 11 Rate Acceleration Catalyzes of a reaction results in rate enhancement not 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) S‡S‡ ES ‡ ES EP Reaction Coordinate Free Energy (delta G) S P

12 Aulani " Biokimia Enzim " Presentasi 1 12 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 ‡ ] S‡S‡ ES ‡ ES EP Reaction Coordinate Free Energy (delta G) S P

13 Aulani " Biokimia Enzim " Presentasi 1 13 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) S‡S‡ ES ‡ ES EP Reaction Coordinate Free Energy (delta G) S P Applications: Inhibitor/drug development based on transition state model Development of catalytic antibodies [rate acceleration up to 10 5 ]

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

15 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 (3) Reactive groups (4) Coenzyme helps (2) (3) (4) (1) CoE + -

16 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

17 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

18 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

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

20 Aulani " Biokimia Enzim " Presentasi 1 20 Essential of Enzyme Kinetics E S + P + Steady State Theory 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. S E E

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

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

23 Aulani " Biokimia Enzim " Presentasi 1 23 The “Active” Enzyme Base catalyzed hydrolysis of an ester: Catalysis is accelerated by altering the poor nucleophile H 2 O into a strong nucleophile OH -

24 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 +

25 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:

26 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]

27 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]

28 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

29 Aulani " Biokimia Enzim " Presentasi 1 29 An Example for Enzyme Kinetics (Invertase) V max KmKm S vovo 1/S 1vo1vo Double reciprocal Direct plot 1) 1) Use predefined amount of Enzyme → E 2) 2) Add substrate in various concentrations→ S (x 3) 3) Measure Product in fixed Time (P/t)→ v o (y 4) 4) (x, y) plot get hyperbolic curve, estimate→ V max 5) 5) When y = 1/2 V max calculate x ([S]) → K m 1 V max - 1 K m 1/2

30 Aulani " Biokimia Enzim " Presentasi 1 30 A Real Example for Enzyme Kinetics Data no Absorbance v (mmole/min) [S] (1) The product was measured by spectroscopy at 600 nm for 0.05 per  mole (2) Reaction time was 10 min VelocitySubstrate ProductDouble reciprocal 1/S1/v →→→→→→→→ v Direct plot Double reciprocal /v /[S] [S]

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

32 Aulani " Biokimia Enzim " Presentasi 1 32 KmKm Enzyme Inhibition (Plots) CompetitiveNon-competitive Uncompetitive Direct Plots Double Reciprocal V max KmKm Km’Km’[S], mM vovo vovo II KmKm V max I Km’Km’ V max ’ V max unchanged K m increased V max decreased K m unchanged Both V max & K m decreased I 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 = K m ’

33 Aulani " Biokimia Enzim " Presentasi 1 33 Ser 195 His 57 Asp 102 H–O–CH 2 O C–O - = Active Ser H–N N CC C H H CH 2 Ser 195 His 57 Asp O–CH 2 O C–O–H = N N–H CC C H H CH 2

34 Aulani " Biokimia Enzim " Presentasi 1 34 pH Influences Chymotrypsin Activity pH Relative Activity

35 Aulani " Biokimia Enzim " Presentasi 1 35 pH Influences Net Charge of Protein + Net Charge of a Protein Buffer pH Isoelectric point, pI

36 Aulani " Biokimia Enzim " Presentasi 1 36 Imidazole on Histidine Is Affected by pH H–N N C C C H H H+H+ pH 6pH 7 + H–N N–H C C C H H Inactive + Ser 195 His 57 Asp 102 H–O–CH 2 O C–O - = H–N N–H CC-H C CH 2 H

37 Aulani " Biokimia Enzim " Presentasi 1 37 Chymotrypsin Produces New Ile16 N-Terminal I16L13Y146 Asp 194 –CH 2 COO - Ile 16 NH 2 – Ile 16 + NH 3 – pH Relative activity pH 9 pH 10 pKa New NH 2 -terminus

38 Aulani " Biokimia Enzim " Presentasi 1 38 New Ile16 N-Terminal Stabilizes Asp194 Asp 102 His 57Ser 195 Asp 194 Gly 193 Ile 16 + NH 3 Catalytic Triad

39 Aulani " Biokimia Enzim " Presentasi 1 39 O (CH 3 ) 2 CH–O– P –O–CH(CH 3 ) 2 F = Chymotrypsin Ser195 Inhibited by DIFP Diisopropyl-fluorophosphate (DIFP) O - …H CH 2 Ser 195 O (CH 3 ) 2 CH–O– P –O–CH(CH 3 ) 2 = O CH 2 Ser 195 XX

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

41 Aulani " Biokimia Enzim " Presentasi 1 41 Chymotrypsin Also Catalyzes Acetate O -C N- H O -C O- Peptide bond Ester bond O CH 3 –C–O– –NO 2 Nitrophenol acetate HO– –NO 2 O CH 3 –C–OH Hartley & Kilby Chymotrypsin+ H 2 O Nitrophenol Acetate No acetate was detected at early stage

42 Aulani " Biokimia Enzim " Presentasi 1 42 O -CO -C Time (sec) Nitrophenol Two-Stage Catalysis of Chymotrypsin O CH 3 –C–O– –NO 2 Nitrophenol acetate OCOC O CH 3 –C HO– –NO 2 + H 2 O O-H C CH 3 COOH Kinetics of reaction Two-phase reaction Acylation Deacylation (slow step)

43 Aulani " Biokimia Enzim " Presentasi 1 43 Extra Negative Charge Was Neutralized O -C N- H O -C-OH NH 2 - -C-C-N-C-C-N-C-C-N- H H E + S O - -C N- HO H O - -C N- HO H

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

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

46 Aulani " Biokimia Enzim " Presentasi 1 46 Classification of Proteases Metal Protease Serine Protease Cysteine Protease Aspartyl Protease Carboxy- peptidase A Chymotrypsin Trypsin Papain Pepsin Renin H57 D102 S195-O - C25-S - H195 D215 D32 H2OH2O Non- specific Non- specific Aromatic Basic Non- polar EDTA EGTA DFP TLCK TPCK PCMB Leupeptin Pepstatin FamilyExample MechanismSpecificityInhibitor E72 H69 Zn 2+H196

47 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 – 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

48 Aulani " Biokimia Enzim " Presentasi 1 48 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) CTP ATP vovo vovo [Substrate] OffOn

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

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

51 Aulani " Biokimia Enzim " Presentasi 1 51


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