Enzymes PDF: Biological Macromolecules and Biocatalysis

VI. Enzymes  What are Enzymes? Enzymes are catalytically active biological macromolecules Most enzymes are globular proteins, however some RNA (ribozymes, and ribosomal RNA) also catalyze reactions Study of enzymatic processes is the oldest field of biochemistry, dating...

VI. Enzymes  What are Enzymes? Enzymes are catalytically active biological macromolecules Most enzymes are globular proteins, however some RNA (ribozymes, and ribosomal RNA) also catalyze reactions Study of enzymatic processes is the oldest field of biochemistry, dating back to late 1700s Study of enzymes has dominated biochemistry in the past and continues to do so  Why Biocatalysis? Higher reaction rates Greater reaction specificity Milder reaction conditions Capacity for regulation - - COO COO NH2 - - O COO Metabolites have OH COO many potential - pathways of - O COO Chorismate COO - decomposition COO OH mutase - OOC O Enzymes make the desired one most NH2 OH favorable  Enzymatic Substrate Selectivity Example: Phenylalanine hydroxylase OH H H - + OOC NH3 - + OOC NH3 H - OOC + NH3 No binding OH HO OH H H Binding but no reaction H NH CH3  Enzyme-Substrate Complex Enzymes act by binding substrates – the non-covalent enzyme substrate complex is known as the Michaelis complex – allows thinking in terms of chemical interactions – allows development of kinetic equations kcat [ E ][ S ] v max[ S ] v  K m  [S ] K m  [S ]  Transition State Theory Slow reactions face significant activation barriers that must be surmounted during the reaction – transition state theory is applicable for catalysis – rate constants and free energies can be related   G   k A exp   RT     G   Without enzyme, slow k A exp   RT   Rate Acceleration The enzyme lowers the activation barrier compared to the uncatalyzed aqueous reaction    G   With enzyme k A exp   RT  Slow Fast  What Do Catalysts Do? Increase the rate of a reaction provide alternative rxn pathway with lower activation energy DG°‡ Cannot change equilibrium position! Cannot change DG°(= change in free energy) of rxn! The higher DG°‡ the slower the rxn! Catalysts lower DG°‡ and speed up rxn. ΔG° = - RT ln K eq  Reaction Rate and Order kf A + B P kr h Forward Rate = kf[A] f [B]g Reverse Rate = kr[P] at equilibrium: forward rate = reverse rate kf = Keq kr k = specific rate constant Σ(f,g) = order of reaction k,f,g measured  Reaction Rate and Order, contd. A + B C + D Rate equation: Rate = k[A]1 [B] 1 Reaction order: first order for A first order for B second order overall (A and B must collide to react) Glycogen n + HPO42- Glucose-1-phosphate + Glycogen n-1 Rate = k[Glycogen] 1[HPO 42-]1 = k[Glycogen][HPO 2- 4 ]  How to Lower G? Enzymes organizes reactive groups into proximity Uncatalyzed bimolecular reactions: two free reactants single restricted transition state conversion is entropically unfavorable Uncatalyzed unimolecular reactions: flexible reactant rigid transition state conversion is entropically unfavorable for flexible reactants Catalyzed reactions: Enzyme uses the binding energy of substrates to organize the reactants to a fairly rigid ES complex Entropy cost is paid during binding Rigid reactant complex transition state conversion is entropically OK Demonstration of entropy effect on reaction rate  How to Lower G? Enzymes bind transition states best The idea was proposed by Linus Pauling in 1946: – enzyme active sites are complimentary to the transition state of the reaction – enzymes bind transition states better than substrates – stronger interactions with the transition state as compared to the ground state lower the activation barrier Largely DH‡ effect  Support for TS Stabilization Stable structural analogs of transition states bind more strongly than reactants Energetics of Biochemical Reactions  How is TS Stabilization Achieved? – acid-base catalysis: give and take protons – covalent catalysis: change reaction paths – metal ion catalysis: use redox cofactors, pKa shifters – electrostatic catalysis: preferential interactions with TS  Acid-base Catalysis: Chemical Example Consider ester hydrolysis: O O O R + H-OH R +H + R + CH 3OH OH O CH3 OH O CH3 Water is a poor nucleophile, and methanol is a poor leaving group Aqueous hydrolysis can be catalyzed either by acids or by bases Enzymes can do acid and base catalysis simultaneously  General Acid-Base Catalysis Example: amide hydrolysis Amino Acids in General Acid-Base catalysis  Covalent Catalysis: Chemical Example O O O O H2O CH3 O - + - + 2 H + H3C O slow H3C H3C O O O O O H3C O CH3 + N + - fast CH3 H3C O.. N The anhydride hydrolysis.. reaction is catalyzed by O pyridine, a better H H nucleophile than water - (pKa=5.5). O.. O + Hydrolysis is accelerated N N CH3 + H3C O - OH because of charge loss in the transition state makes H + pyridine a good leaving group.  Covalent Catalysis: In Enzymes Proteases and peptidases – chymotrypsin, elastase, subtilisin – reactive serine nucleophile Some aldehyde dehydrogenases – glyceraldehyde-3phosphate dehydrogenase – reactive thiolate nucleophile Aldolases and decarboxylases – amine nucleophile Dehalogenases – carboxylate nucleophile NH2 - O HO S - O O O O O N N N N Chymotrypsin Active site Chymotrypsin Mechanism Step 1: Substrate Binding Chymotrypsin Mechanism Step 2: Nucleophilic Attack Chymotrypsin Mechanism Step 3: Substrate Cleavage Chymotrypsin Mechanism Step 4: Water Comes In Chymotrypsin Mechanism Step 5: Water Attacks Chymotrypsin Mechanism Step 6: Break-off from the Enzyme Chymotrypsin Mechanism Step 7: Product Dissociates  What is Enzyme Kinetics? Kinetics is the study of the rate at which compounds react Rate of enzymatic reaction is affected by – Enzyme – Substrate – Effectors – Temperature  Why Study Enzyme Kinetics? Quantitative description of biocatalysis Determine the order of binding of substrates Elucidate acid-base catalysis Understand catalytic mechanism Find effective inhibitors Understand regulation of activity How to Do Kinetic Measurements  Effect of Substrate Concentration Vmax[ S ] kcat [ E ][ S ] Ideal Rate:v  K m  [S ] v K m  [S ] Deviations due to: Vmax=kcat[E] – Limitation of measurements – Substrate inhibition – Substrate prep contains inhibitors – Enzyme prep contains inhibitors  Determination of Kinetic Parameters Nonlinear Michaelis-Menten plot should be used to calculate parameters Km and Vmax Vmax [S] V= Km+ [S] Linearized double-reciprocal plot (Lineweaver- Burk) is good for analysis of two-substrate data or inhibition 1/V = 1/Vmax + Km/Vmax x 1/[S]  Vmax [S] V= Km+ [S] How to Determine KM and Vmax more Accurately? V max [S] V= (M-M equation for hyperbola) KM + [S] 1 KM + [S] KM + [S] = = V V max [S] V max [S] V max [S] 1 KM 1 = + V V max [S] V max 1 = KM + 1 V Vmax [S] Vmax Lineweaver-Burk Equation  The Lineweaver-Burk Equation 1 KM 1 1 = + V init V max [S] V max y = m x + b slope y-intercept Plot 1/Vinit versus 1/[S]  straight line Variables: 1/Vinit and 1/[S] ; can be measured Slope = KM/Vmax ; y-intercept = 1/Vmax 1/V = 1/Vmax + Km/Vmax x 1/[S]  Km, Vmax, kcat, and kcat/Km High Km low affinity (unit M or mM) hexokinase substrate KM ATP 0.4 mM Glucose 0.05 mM Fructose 1.5 mM Cabonic anhydrolase HCO3- 2.6 mM High Vmax high catalytic rate (M.s-1) Turnover number kcat = Vmax/[E] (s-1) (e.g. 10 s-1) Catalytic efficiency kcat/Km (M-1.s-1)  Calculations Happyase SAD  HAPPY 1) kcat=600 s-1, [SAD]=40 uM, [E]=20 nM, V0=9.6 uM.s-1, Km=? Vmax [S] Vmax [S] V0 = Km = - [S] =(kcat[E]/V0-1)[S] Km+ [S] V0 =(600 s-1x 0.02 uM/9.6 uM.s-1-1)x40 uM = 10 uM 2) Same E, [E]=10 nM, V0=3.0 uM.s-1, [S]=? V0 Km V0 Km 3 uM.s-1 x 10 uM [S] = = = Vmax- V0 kcat[E] - V0 600 s-1 x 0.01 uM -3 uM.s-1 = 10 uM The Lineweaver-Burk Plot  Example Calculation [S] (mM) V (mM sec-1) 2.5 0.024 5.0 0.036 10.0 0.053 15.0 0.060 -1 20.0 0.061 [S-1] (mM-1) V-1 (mM sec-1)-1 0.400 41.667 0.200 27.778 0.100 18.868 0.067 16.667 0.050 15.625  Plot the Converted Data and Calculate Slope: Low [S] need to use best fit line! -1 Infinite [S] Vmax-1 = 12 Vmax = 0.083 mM sec-1 * High [S] [S-1] (mM-1) V-1 (mM sec-1)-1 0.400 41.667 -KM-1 = -0.155 0.200 27.778 KM = 6.45 mM 0.100 18.868 0.067 16.667 0.050 15.625 -1  How to Calculate the Slope? Slope = rise over run need to use best fit line, NOT individual data points! -1 Rise = 30-20 = 10 Slope = KM/Vmax = 10/0.13 = 76.92 * -1 Run = 0.24-0.11 = 0.13  Two-substrate Reactions Kinetic mechanism: the order of binding of substrates and release of products When two or more reactants are involved, enzyme kinetics allows to distinguish between different kinetic mechanisms – Sequential mechanism – Ping-Pong mechanism  Enzyme Inhibition Inhibitors are compounds that decrease enzyme’s activity Irreversible inhibitors (inactivators) react with the enzyme - one inhibitor molecule can permanently shut off one enzyme molecule - they are often powerful toxins but also may be used as drugs Reversible inhibitors bind to, and can dissociate from the enzyme - they are often structural analogs of substrates or products - they are often used as drugs to slow down a specific enzyme Reversible inhibitor can bind: – To the free enzyme and prevent the binding of the substrate – To the enzyme-substrate complex and prevent the reaction Same binding site Vmax [S] V0 = αKm+ [S] α = 1 + [I]/KI Competitive Inhibition Plots Competitive  Different binding sites Vmax [S] V0 = Km+ α’[S] α’ = 1 + [I]/KI’ Noncompetitive Inhibition Plots Uncompetitive  Different binding sites independent Vmax [S] V0 = αKm+ α’[S] Mixed  VAPmax [S] V0 = KAPm+ [S]  Enzyme Inhibitors and Medicine Penicillin (Alexandra Fleming, 1928) – Transpeptidase, catalyzing the formation of peptidyglycan – Penicillin forms a covalent bond with transpeptidase Protease inhibitors – HIV expression polyprotein (inactive)active viral proteins by an HIV protease – Indinavir, Nelfinavir, Lopinavir containing C-C(OH)-C as tetrahedral intermediate mimics and phenyl group for hydrophobic binding – Irreversible inhibitors, high affinity, KI nM, very effective inhibitors  Classification of Reversible Inhibitors Reversible inhibitor can bind: – To the free enzyme and prevent the binding of the substrate – To the enzyme-substrate complex and prevent the reaction Feedback inhibition  Chapter 6: Summary In this chapter, we learned about: why nature needs enzyme catalysis how enzymes can accelerate chemical reactions how chymotrypsin breaks down peptide bonds how to perform and analyze kinetic studies how to characterize enzyme inhibitors  Chapter 6 Important Concepts and Principles Enzyme definition and general features b. Transition state theory, activation energy, and enzyme catalysis. Transition state analog. General principles of TS stabilization by enzymes. Acid-base catalysis and covalent catalysis Chymotrypsin mechanism of peptide hydrolysis. Enzyme kinetics, factors involved. Michaelis-Menton and Lineweaver-Burk equations & plots Definition and meaning of kcat, Vmax, Km, and kcat/Km Enzyme kinetics calculation. Enzyme efficiency comparison  Chapter 6 Important Concepts and Principles Enzyme inhibitors and different types m. Competivive, uncompetitive, mixed inhibitors – model, formula, Ki, LB plots. Enzyme inhibitors and medicine