Você está na página 1de 28

4:211 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 tKwMeand
e n K S?

Check Fersht Ch. 3 - A.3.b. (pp 107-108) for opposite a


actually more general case.
Eadie - Hofstee plot
v
v = Vmax- KM
[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
KA KB kp
E+A EA + B EAB 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
V (s-1)

9.5
30
14
19
20

10

0 1 2 3 4 5
+
[NAD ] (mM)

0.3
]/V (mM*min.)

0.2
[NAD +

0.1

0
-1 0 1 2 3 4 5
+
[NAD ] (mM)
Transient kinetics: flow apparatuses
Stoped flow

Quenched flow

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

Proton transfer rates involving imidazole (PKa=6.95)

Enymes for which kcat /KM is close to be diffusion controlled

Você também pode gostar