BL4820 Biochemistry Techniques

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BL4820 BASIC BIOCHEMICAL TECHNIQUES -- Lecture 3 - Enzyme Kinetics -- Expt 2 Part B

5. Calculation of the Ki for Competitive and Non-Competitive Inhibitor

The strength of the complex formed between the enzyme and the inhibitor can be calculated from the kinetics constants obtained in the analysis of steady state kinetics:

Table of Equations for Calculating Inhibitor Binding Constants (Ki values).

The symbols Km and Vmax have the same meaning here as in the standard kinetic analysis, while in the inhibited reactions, Km' represents the "Km" obtained in the graphical solution of the data collected in the presence of a competitive inhibitor and Km' will always be greater than Km. Vmax' is obtained from the graphical solution of the data collected in the presence of the noncompetitive inhibitor and will always be less than Vmax. The inhibitors of both types are used at known concentrations ([I]) and these must be used to find the strength of the binding of the inhibitor to the enzyme (Ki) and the Ki has the same units of concentration as does the inhibitor. So if [I] = 1 mM, then the units of Ki will be mM. So the degree to which the inhibitor effects the kinetics of the enzyme depends on both the strength of its complex with the enzyme, with a low Ki indicating strong binding of the inhibitor to the enzyme, and the concentration of the inhibitor added to the kinetic analysis. When you make your lab report, calculate the Ki constants for Pi and F- and compare them. The one with the lower Ki is the stronger inhibitor.

Which is the stronger inhibitor of acid phosphatase, Pi or F^-, according to your kinetic data?

You can in another sense compare the Ki constants to the Km for substrate, which is also expressed as a concentration, and make a general judgment as to whether the strength of the Michaelis complex (sort of the affinity of the enzyme for the substrate) is greater than the strength of the complex of inhibitor and enzyme. Since the competitive inhibitor binds at the same site as the substrate (especially in the case of the phosphatase you are studying since the competitive inhibitor being used is the product of the reaction), how does the Ki for Pi compare to the Km for pNPP? In another sense, the noncompetitive inhibitor, which apparently binds to the enzyme at a place other than the substrate binding site, will generally have a Ki with less meaningful relationship to the Km of the enzyme.

Back To: Lecture 3 - Enzyme Kinetics

Copyright ©1996, 1997, 1998, 1999, 2000, 2001 Wilbur H. Campbell, All Rights Reserved; wcampbel@mtu.edu

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