1 / 33

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:

alicia
Télécharger la présentation

Enzyme Kinetics

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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)

More Related