BioChem 330 - Course Outline Catalysis

BioChem 330 - Course Outline October 13,18, 2011 • Bio-molecular Structure/Function (I cont’d) – NUCLEIC ACID • DNA sequence and structure • Protein/nucleic acid interactions – CARBOHYDRATES • Sugars - mono and disaccharides • Polysaccharides • Glycerides and glycerol – FATS AND LIPIDS • Chemistry and nomenclature for fatty acids • Saturated and unsaturated fatty acids • Fluid mosaic model of membrane structure BioChem 330 - Course Outline • Metabolism and Bioenergetics (II) – ENZYME CATALYSIS: • kinetic constants kcat, Km • Catalytic strategies, the serine proteases – CATABOLISM (breakdown) • Carbohydrates – – – – Glycolysis Tricarboxylic Acid Cycle Electron Transport Chemiosmosis and ATPase • Fatty acids and amino acids REACTION COORDINATE DIAGRAMS • Intermediates: – A, B and C are intermediates in the conversion of S to P. Intermediates can accumulate and reach steady states in concentration. • TS Complexes: S k1 k2 k-1 -2 k3 k4 A k B  CP k k -3 -4 Reaction Coordinate Diagram can be reconstructed once all the forward and reverse rate constants are measured at a particular T. – S/A‡, A/B‡, B/C‡, and C/P‡ are transition state complexes which have a fleeting existence and can only be poorly defined. REACTION COORDINATE DIAGRAMS S k1 k2 k-1 -2 k3 k4 A k B  CP k k -3 -4 • • – The rate or velocity, V, is limited by the rate determining step; that step with the largest barrier, or smallest rate constant, here A→B, k2 – V depends on the combined rates of all the elementary steps up to the slowest step, which determines [A]. – Conversion A →B k2 = 103s-1 – Activation Barrier: – DG‡ = 17.6 - 1.36 log k = 17.6 - 1.36 log 103 = 13.5 kcal/mole at 25.0oC What is the order of enzyme reaction w.r. to [S]? Exp. measurement Calculated from data • Biological conditions of small [E] compared to [S]. • Reaction is first order with respect to [S] at low concentrations of [S], then zero order at high [S] • Can only be explained by presence of specific [ES] complex. What is the order of the reaction with respect to [E]? Experimental measurement Calculated from data • Here, [S] is held constant and Rate is measured as a function of enzyme concentration (but still at concentrations of [E] much lower than [S] • Rate is first order in [E]. • Whatever rate expression you propose, it must have the three characteristics, first order in [S] at low [S], zero order in [S] at high [S] and first order in E. Proposed Michaelis Menten Mechanism • E+S k1  k -1 k2 [E] << [S] , [S] = [St] ES ------> E + P • Assume ES reaches a steady state with E and S – Rate of formation of ES equals the rate of breakdown – k1[E][S] = k-1 [ES] + k2 [ES] = (k-1 + k2)[ES] – [ES] = k1[E][S] /(k-1 + k2) = [E][S] / Km Km = (k-1 + k2) /k1 • Assume k2 is the catalytic step: product dissociates rapidly – Velocity = k2 [ES] – [ES] =[E][S] /Km – [ES] =(([Et] - [ES])[St]) /Km [E] = [Et] - [ES] [ES] = [Et] [St] • Solve this for ES • Plug into Velocity equation above Km + [St] • Velocity = k2 [Et] [St] = kcat [Et] [St] = Vmax [St] • Km + [St] Km + [St] Km + [St] Define: Vmax= kcat[Et] Michaelis Menten Kinetics • Velocity = Vmax [St] • Km + [St] • When [S] is small, the reaction is first order. • When [S] is large, the reaction is zero order in [S], velocity is at Vmax • Reaction is always first order in [E] • When [S] = Km, V= ½Vmax ES Complex* Michaelis Complex • Lock and Key Model Induced Fit Model • Km measures the affinity of an enzyme for a particular substrate (often an enzyme can bind and catalyze reaction on a family of closely related substrates) • higher Km, lower binding, Km is like a dissociation constant Graphical Means of Extracting M2 Parameters Velocity = kcat [Et] [St] = Vmax [St] Km + [St] Km + [St]     Hyperbolic Curve Vmax = asymptotic max. kcat = Vmax/ [Et] Km = [S] at ½ Vmax Lineweaver-Burk Plot Vmax = 1/Yintercept kcat = Vmax/ [Et] Km = - 1/Xintercept Lineweaver Burk Plots Since Vmax = kcat [ET], once you have Vmax, you can calculate kcat    These plots, known as double reciprocal plots, are a much preferred way of plotting data for an enzyme catalyzed reaction, though this idealizes error. Km is derived from the -1/X intercept Vmax is derived from the 1/Y intercept Reversible Enzyme Inhibition   Competitive inhibitors slow down a reaction by competing with substrate for the active site, kcat not affected but Km appears to be higher (appears to be worse binding) Noncompetitive inhibitors slow down a reaction by binding at a remote site and slowing down kcat, Km not affected. How to Distinguish Types of Inhibition  Competitive and Noncompetitive Inhibition can be distinguished by how they affect the double reciprocal plots Meaning of Michaelis Constants  I. kcat (turnover number).... the number of substrate molecules converted into product molecules by an enzyme molecule in a unit of time when the enzyme is fully saturated with substrate. Turnover number is calculated from kcat =Vmax/Et  Has units of s-1 (like a first order rate constant)  May or may not be a microscopic rate constant, in M2 mechanism, kcat = k2  Range of about 1-107 s-1  The turnover number of most enzymes falls between 1 and 10,000 substrates converted to product per enzyme molecule per sec.  I. kcat (turnover number) continued Turnover numbers Maximum turnover # of some enzymes kcat (per s) reaction time carbonic anhydrase 600,000 1.7 us 3-ketosteroid isomerase 280,000 3.6 us Acetylcholinesterase 25,000 40. us Penicillinase 2,000 500. us Chymotrypsin 100 10. ms DNA polymerase I 15 67. ms Tryptophan Synthase 2 500. ms Lysozyme 0.5 2 s NO MICROSCOPIC RATE CONSTANT CAN BE SLOWER THAN kcat kcat cannot be greater than any first order rate constant along the forward reaction pathway, and thus helps to set a lower limit on all of the microscopic rate constants. I. kcat (turnover number) continued • What is a microscopic rate constant? • NO MICROSCOPIC RATE CONSTANT CAN BE SLOWER THAN kcat k1 k2 k3 k-1 k-2 k-3 kcat • E + S ↔ ES1 ↔ES2 ↔ ES3------> E + P microscopic rate constants • kcat cannot be greater than any first order rate constant along the forward reaction pathway, and thus helps to set a lower limit on all of the microscopic rate constants.  II. Km, the Michaelis constant  experimentally measured from [S] at ½ Vmax  Units of M or mM or μM  represents dissociation of Michaelis complex and is either a true or apparent dissociation equilibrium constant  Km = [E] [S] / ∑[ESn]  Range 10-7 to 10-3 M  Enzyme Substrate Km (mM)       Chymotrypsin Lysozyme b-Galactosidase Thr deaminase Carbonic Anhydrase Pyruvate carboxylase – – acetyl-tryptophanamide hexa-N-acetylglucosamine Lactose Threonine CO2 Pyruvate HCO3ATP 5000 6 4000 5000 8000 400 1000 60   II. Km, the Michaelis constant (cont’d) It is informative to compare the natural substrate concentration and Km, [S]/Km  If  [S]/Km < 1; [S] 1; [S] > Km ; the enzyme is saturated with substrate and the system is working at full capacity, but NOT sensitive to changes in substrate concentration. The [S]/Km ratio is typically between 0.01 and 1.0. This can be interpreted as most systems not working at full capacity, so that they function with a built in throttle to rapidly accelerate the rate should there be a sudden biological demand.  III. Kinetic Perfection in Enzymic Catalysis, The kcat/Km criterion  Velocity  = kcat [Et] [St] Km + [St]   When enzymes are not saturated with substrate, V    = (kcat/Km) [Et] [S] Graphically (kcat/Km) [Et] is the slope of V vs [S] at low [S] kcat/Km has units of M-1s-1 kcat/Km is an apparent second order rate constant looks at free [E] with [S] and is really of interest when studying enzyme-substrate specificity     III. Kinetic Perfection in Enzymic Catalysis, The kcat/Km criterion kcat/Km is a critical parameter when assessing enzyme specificity for competing substrates, if kcat/Km increases, the specificity increases. Some enzymes have kcat/Km of 107 or 108 M-1s-1, which is amazing specificity! (diffusion controlled second order rate constants are about this value) Let’s look at the effects of small structural changes in the substrate on the kinetic parameters for chymotrypsin catalyzed amide hydrolysis: III. Kinetic Perfection in Enzymic Catalysis, The kcat/Km criterion A Family of Serine Proteases  Substrate specificity pocket helps chymotrypsin choose the right substrate   large nonpolar channel into which side group can be place adjacent to scissile bond (bond to be cut by enzyme) Other serine proteases have other specificity pockets Lys, Arg for Trypsin (Asp in bottom of oxyanion hole  Ala, Val for Elastase  Proteases: Enzymes that Hydrolyze Peptide Bonds  Catalytic Triad in Serine Proteases: Serine 195 Nucleophile  His 57, neutral, acts as a proton shuttle  Asp 102, activates His through H bonding  Proteases: Enzymes that Hydrolyze Peptide Bonds  How the catalytic triad works Ser is activated by His, to which it transfers a H when it attacks the carbonyl of the substrate  His holds onto H from serine transiently, His+ stabilized by Asp- in acyl enzyme intermediate  Catalytic Mechanism for Trypsin   Phase I E + S = ES --> [ES]tt --> EA + P1 (amino) Catalytic Mechanism for Trypsin   Phase II E A --> [EA]tt --> E + P2 (carboxylate) Proteases:Enzymes that Hydrolyze Peptide Bonds       Amide Bond Hydrolysis R1 O R2 R1 O ---CH-C- NH - CH- + H2O → R2 ---CH-C-OH + HNH-CH-P2 P1 k2 k3 E + S = ES → EA + P1 → E + P2 For amide bond hydrolysis, formation of EA is rate limiting, k2>k3