Steady State Kinetics

10/8/98


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NOTICE: This material is copyrighted and all right are reserved. (c) 1998 J. E. Wampler
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Table of Contents

{1} Steady State Kinetics

{2} What Defines a Kinetic Model?

{3} Example: Glucokinase (GK) reaction

{4} Kinetic mechanism depends on order of substrate binding.

{5} So, one of several models might be:

{6} SHORT HAND NOTATION (The Cleland Diagram):

{7} The second focus, given a model, is the rate constants

{8} More about rates & rate constants

{9} More about rates & rate constants...

{10} Another way to represent the overall reaction scheme is the King-Altman diagram:

{11} Steady State Kinetics

{12} Single Substrate/Product Models

{13} This is the Michaelis-Menten Model, MODEL #1

{14} A variety of other models also give the Michaelis-Menten Equation

{15} MODEL #2: E + S ES E + P Continued:

{16} E + S ES E + P Continued:

{17} E + S ES E + P Continued:

{18} E + S ES E + P Continued:

{19} E + S ES E + P Continued:

{20} E + S ES E + P Continued:

{21} One more model, MODEL #3:

{22} Steady State continued:

{23} Other models which give the Michaelis-Menten Equation:

{24} Finally, a more realistic model:

{25} What experiment can we do to distinguish between these models?

{26} Steady State Kinetics

{27} Back to the glucokinase reaction...

{28} How do we get a rate equation from these models?

{29} Let A = 1st substrate, B = 2nd substrate, P = 1st product and Q = last product

{30} where N's and D's are sums and products of rate constants.

{31} Working with complex rate equations:

{32} A further limitation makes this equation behave like the Michaelis-Menten equation...

{33} Any equation of this form...

{34} Analysis gives apparent kinetic constants...

{35} In general the macroscopic constants are related to the coefficients of the rate equation as follows:

{36} How do these models differ?

{37} 2) With the Steady State derivation, the equation is no longer symmetrical in A and B terms (or P and Q).

{38} Rearranged...

{39} When B is the variable substrate,

{40} What experiments can we do to distinquish between these models?

{41} So, what about the Glucokinase reaction?

{42} Steady State Kinetics

{43} More on inhibitors

{44} More on inhibitors...

{45} More on inhibitors...

{46} More on inhibitors...

{47} Steady State Kinetics

{48} More about Cleland Nomenclature and Reaction Schemes

{49} Reaction schemes...

{50} Cleland Nomenclature (see Biochim. Biophys. Acta 67, 104. 1963)

{51} Cleland Nomenclature Continued:

{52} Cleland Nomenclature Continued:

{53} Cleland Nomenclature Continued:

{54} Cleland Nomenclature Continued:

{55} What about King-Altman Diagrams?

{56} Steady State Kinetics

{57} Defining other Bi Substrate, Bi Product models:

{58} What about Random Bi Bi?

{59} Changes in the Model with Equilibrium Steps:

{60} Obviously, all models don't reduce to the M-M form.

{61} 4) Interconvertable, Active Enzyme Forms.....

{62} 5. Substrate Inhibition....

{63} 5. Substrate Inhibition....

{64} Steady State Kinetics

{65} Multisite Enzymes can give either M-M Kinetics or higher order behavior

{66} Multisite Enzymes...

{67} Multisite Enzymes...

{68} Multisite Enzymes...

{69} The Symmetry Model (Monod, Wyman & Changeux):

{70} The Symmetry Model...

{71} The Symmetry Model...

{72} The Induced Fit Model (Koshland, Nemethy & Filmer):

{73} The Induced Fit Model...

{74} Multisite Enzymes…Summary

{75} Steady State Kinetics

{76} More about Cooperativity

{77} Some Examples:

{78} Cooperativity with oxygen binding proteins:

Author: John E. Wampler

Email: wampler@bchiris.bmb.uga.edu

Home Page: http://bmbiris.bmb.uga.edu/wampler/8010

Assignment:

Garrett & Grisham, Chapters 14 and 15 including Appendex to Chapter 15
References:

A. Cornish-Bowden (1979) Fundamentals of Enzyme Kinetics, Butterworths, London.

A. Fersht (1985) Enzyme Structure and Mechanism, W. H. Freeman and Co., New York.

A. R. Schulz (1994) Enzyme Kinetics, Cambridge University Press, Cambridge.