Quiz #3 • Define Enzyme Classes • Systematic naming – Given a reaction (including names) – Use subclass designation if appropriate • Catalytic mechanisms – Define – How does a given amino acid participate? • Discuss protonation state and reaction. Enzyme Kinetics The study of the rates at which enzyme-catalyzed reactions occur. Value of Enzyme Kinetics • Quantitation of Enzyme Activity – Purification • Competing Substrates and Inhibitors – Suggest physiological functions – Suggest regulatory mechanisms • Response to Different Conditions – Reaction mechanism Kinetic Data plus Structure and Mechanism • Clues to an enzyme’s biological function • Ways to modify an enzyme for therapeutic purposes Enzyme-Catalyzed Reactions are Reversible (Accelerate Attainment of Equilibrium) Elementary Reactions A ——> P Reaction Intermediates A ——> I1 ——> I2 ——> P Two-Step Reactions (Rate Determining Step) A k1 I k2 P Chemical Kinetics • Can reveal the number of reacting species • Can reveal the rate of their interactions • Are described by Rate Equations Rate Equations • Reaction Order or Molecularity: number of molecules participating in a reaction • Velocity (V): molar per second (M•s–1) • Rate Constant (k): proportional to frequency of interaction between reactants Rate Equations (General Form) aA + bB ——> P V = k[A]a[B]b k = rate constant Order of Reaction = a + b Reaction Order (Reaction Molecularity) • First-order or unimolecular • Second-order or bimolecular • Third-order very rare • 4th-order are unknown Rates of Reactions First-Order Reactions A P d[A] d[P] v = – = = k[A] dt dt k = 1st order rate constant (s–1) Time Course of Reaction Equilibrium A or P A ——> P PB A or B t1/2 (half-life) AA Time Kinetics of Reactions First-Order Reaction Substrate [S] kF kB Product [P] ("Reactant") V = kF[S] – kB[P] kF[S] = kB[P] at equilibrium Initial Velocity (VO) Substrate [S] kF kB Product [P] ("Reactant") V = kF[S] – kB[P] V = kF[S] – kB[P] Initial Velocity (Vo) ([P] = 0) kF[S] = kB[P] at equilibrium Vo = kF[S] Uncatalyzed Reaction • Vo slope = kF [S] Vo = kF [S] Enzyme Catalysis Vm ax VO not proportional to [S] Vo Vo proportional to [S] [S] Hyperbolic Relationship Michaelis-Menten Formulation Enzyme Kinetics Experiment: (1) Mix enzyme + substrate (2) Record rate of product formation as a function of time (the velocity of reaction) (3) Plot initial velocity versus substrate concentration. (4) Change substrate concentration and repeat Postulated Pathway of Enzyme Catalysis: Formation of Enzyme-Substrate Complex E + S k1 k-1 Binding E—S k2 Catalysis E + P Assumptions: Initial Velocities Catalysis is rate limiting E + S k1 k-1 Binding E—S k2 Catalysis E + P (rate limiting) Vo = k2[ES] Assumptions • Binding equilibrium (k–1>>k2) E + S k1 k-1 Binding E—S k2 E + P Catalysis • Steady state (d[ES]dt = 0) Assumptions • Binding equilibrium (k–1>>k2) E + S k1 k-1 Binding E—S k2 E + P Catalysis • Steady state (d[ES]dt = 0) Additionally • [s]>>>>>>>>>[E-S] – [S] doesn’t change during initial velocity • [ET] = [EF] + [E-S] Progress Curves (M-M Enzyme) Steady-State Very short Michaelis-Menten Equation VO= Vmax[S] Km + [S] Assumes All Enzyme Molecules are Catalytically Active Michaelis Constant Km = k-1 + k2 k1 KM is the substrate concentration at which the reaction velocity is half-maximal High affinity = low Km; Low Affinity = high Km Michaelis-Menton Enzyme Kinetics Catalytic Constant (Turnover Number) Kcat = Vmax [E]T # of substrate converted to product = Unit of time # of enzyme Vmax = kcat X # of enzymes # can be any units (molecules, moles, μmoles) But all must be the same units Catalytic Efficiency • kcat/KM – Limited by diffusion (108 to 109 M-1s-1) Lineweaver-Burk Formulation 1 Vo = KM 1 Vmax [S] + 1 Vmax Lineweaver-Burk Plot Eadie-Hofstee Formulation Vo = –KM Vo [S] + Vmax Eadie-Hofstee Plot Vo Vmax Slope = –Km Vmax Km Vo S A certain enzyme catalyzes the conversion of 100 μmoles of A to B in one second, when the concentration of A is 20 mM. It catalyzes the conversion of 350 μmoles of A to B in one second when the concentration of A is either 500 mM or 550 mM. -Calculate Km -Draw the Michaelis-menton curve and Lineweaver-Burk plot for this enzyme -Can you calculate turnover #?