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Enzyme Kinetics

Enzyme Kinetics. Chapter 6. Kinetics. Study of rxn rates, changes with changes in experimental conditions Simplest rxn: S  P Rate meas’d by V = velocity (M/sec) Depends on k, [S]. Michaelis-Menten Kinetics. Gen’l theory rxn rate w/ enzymatic catalysis Add E, ES to rxn:

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Enzyme Kinetics

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  1. Enzyme Kinetics Chapter 6

  2. Kinetics • Study of rxn rates, changes with changes in experimental conditions • Simplest rxn: S  P • Rate meas’d by V = velocity (M/sec) • Depends on k, [S]

  3. Michaelis-Menten Kinetics • Gen’l theory rxn rate w/ enzymatic catalysis • Add E, ES to rxn: E + S  ES  E + P • Assume little reverse rxn E + P  ES So E + S  ES  E + P • Assign rate constants k1, k-1, k2

  4. Assume: Vo condition -- [S] >>> [E] • Since S used up during rxn, can’t be limiting • Assume: All E goes to ES • Assume: Fixed amt enzyme • If all E  ES, will see max rate of P formed • At steady state rate form’n ES = rate breakdown ES

  5. Exper’l Findings: • As incr [S], V incr’s linearly up to some max V • At max V, little V incr regardless of [S] added

  6. M-M Relates [E], [S], [P]  Exper’ly Provable Variables • New constant: KM = (k2 + k-1) / k1 • M-M eq’n: Vo = (Vmax [S]) / (KM + [S])

  7. Quantitative relationship between • Initial velocity • Max rate of rxn • Initial [S]

  8. Exper’l Definition of KM • At ½ Vmax (substitute ½ Vmax for Vo) • Divide by Vmax • Solve for KM • KM = [S] • So when Vo = ½ Vmax , KM = [S]

  9. Difficult to Determine Variables from M-M Plot • Hard to measure small changes in V • Use double reciprocal plot  straight line • Lineweaver-Burk (Box 6-1)

  10. KM • [S] at which ½ enz active sites filled • Related to rate constants • In living cells, value close to [S] for that E • Commonly enz active sites NOT saturated w/ S

  11. May describe affinity of E for S ONLY if k-1 >>> k2 • Right half of rxn equation negligible • KM = k-1 / k1 • Describes rate form’n, breakdown of ES • Considered dissociation constant of ES complex • Here, KM value indicates strength of binding E-S • In real life, system is more complex

  12. Other Kinetics Variables • Turnover # • kcat • # S molecules converted  P by 1 enz molecule per unit time • Use when enz is fully sat’d w/ S

  13. Comparisons of Catalytic Abilities • Optimum KM, kcat values for each E • Use ratio to compare catalytic efficiencies • Max efficiency at kcat / KM = 107– 108 M-1 sec-1 • Velocity limited by E encounters w/ S • Called Diffusion Controlled Limit

  14. Kinetics When>1 Substrate • Random order = E can accept either S1 or S2 first • Ordered mechanism = E must accept S1 first, before S2 can bind • Double displacement (or ping-pong) = S1 must bind and P1 must be released before S2 can bind and P2 is released

  15. Inhibition • Used by cell to control catalysis in metabolic pathways • Drugs, toxins alter catalysis by inhib’n • Used as tools to study mechanisms • Irreversible • Reversible • Includes competitive, noncompetitive, uncompetitive

  16. Irreversible Inhibition • Inhibitor binds tightly to enz • Dissociates slowly or not at all • Book example: DIFP • Includes suicide substrate inhibitors

  17. Reversible Inhibition • Inhibitor may bind at active site or some distal site • Binding reversible • Temporarily inhibits E, S binding or proper rxn • Can calculate KI

  18. Competitive • “Appear as S” • Bind active site • So compete w/ S for active site • Overcome w/ incr’d [S] • Affects KM, not Vmax

  19. Reversible Inhib’n (cont’d) • Uncompetitive • Binds only when S already bound (so ES complex) • Bind at site away from active site • Causes conform’l change, E inactivated • Not overcome w/ incr’d [S] • Affects both KM, Vmax • Common when S1 + S2

  20. Reversible Inhib’n (cont’d) • Noncompetitive (Mixed) • When S bound or not • Bind at site away from active site • Conform’l change in E • E inact’d when I bound • Decr’d E avail for binding S, rxn catalysis • Not overcome w/ incr’d [S] • Affects both KM, Vmax • Common when S1 + S2

  21. Effect of pH on Catalysis • Optimum pH where max activity • Aa’s impt to catalysis must maintain partic ionization • Aa’s in other parts of enz impt to maintain folding, structure must also maintain partic ionization • Can predict impt aa’s by activity changes at different pH’s (use pKa info)

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