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Introduction to enzyme kinetics

10/3/01

The significance of enzyme kinetics Derivatisation of rapid equilibrium equations The steady state assumption and Derivation of the Briggs-Haldane equation Linear transformations and linear plots and their applications Time integrated equation Inhibition kinetics - techniques and uses Nomenclature Rapid kinetics - experimental examples Limits of kinetic rate constants and the rate determining step Useful kinetic shortcuts

References 1. Fersht, Ch. 3. and 4 2. Cleland, Meths Enzymol. 87, 390-405 (1982). pH analysis. The remainder of this volume has considerable useful information for the practicing kineticist. 3. Cleland, adv. Enzymol. 45, 273-387 (1977). Most of what the normal kineticist will ever have to know is to be found here. 4. Segel, "Enzyme Kinetics" (1975), Wiley. Very comprehensive book. 5. King, E.L & Altman C. J. Phys. Chem. 60, 1375-1378 (1956). The King-Altman method for driving kinetic equations. 6. Cleland, Biochemistry 14, 3220 (1975). The net rate constant method for the analysis of linear mechanisms. 7. Cha, J. Biol. Chem. 243, 820-825 (1968). Rapid equilibrium simplification of King Altman procedure. 8. Frieden, Trends in Biochemical Sciences, 4, 181-182.(1994) Numerical integration of rate equations by computer. 9. Northrop, D.B. j. Chem. Ed. 75, 1153-1157 (1998). On the meaning of KM and V/K.

Derivatisation of rapid equilibrium equations

k 2 < < k -1

v=

f ([S])

The steady state assumption

Derivation of the Briggs-Haldane equation


The steady state assumption

W h a t a r e t h e r e l a t i o n s b e tK w e n K S? Me and Check Fersht Ch. 3 - A.3.b. (pp 107-108) for opposite a actually more general case.

Eadie - Hofstee plot


v = Vmax- KM v [S]

What is the principal advantage of this plot? The integrated equation

Competitive inhibition

Graphic presentations

Dixon plot

Non-competitive inhibition

Graphic presentation

Uncompetitive inhibition

Summary: Eadie-Hofstee vs. Lineweaver-Burk plots

Mixed inhibition

Ordered Bi-reactants reaction


E+A
KA KB

EA + B

EAB

kp

E+P

Rapid equilibrium random Bi-reactant reaction

Ping - Pong mechanism

Alternative schematic presentations

Application

Life can be more complex than that

Initial Velocity Measurements


3D Matrix Fitting
v = Vmax[A][B]/(KiaKB+KA[B]+KB[A]+[A][B])
60 50

Exp. v
40

2.0 mM BnOH 4.5 9.5 14 19

V (s-1)

30 20

10 0

2
+

[NAD ] (mM)
0.3

]/V (mM*min.)

0.2

[NAD +

0.1

-1

2
+

[NAD ] (mM)

Transient kinetics: flow apparatuses


Stoped flow

Quenched flow

Pulse flow

Proton transfer rate constants (25C, s -1 M-1)

Proton transfer rates involving imidazole (PKa=6.95)

Enymes for which kcat /K M is close to be diffusion controlled

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