Humboldt State University ® Department of Chemistry

Richard A. Paselk

Chem 431	Biochemistry	Fall 2001
Lecture Notes:: 15 October	© R. Paselk 2001
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Enzyme Kinetics

ENZYME KINETICS AND INHIBITION

Three major types of inhibition:

• Competitive inhibition
• Noncompetitive inhibition
• Uncompetitive inhibition
  

Competitive Inhibition: S & I are mutually exclusive, E can bind to one OR the other.

Plots:

We can model this inhibition with chemical equations, keeping in mind that S & I are mutually exclusive, E can bind to one OR the other:

Model: (p 316); and: ; where (Table 12.2, p 340).

Classically assume binding to same site, but other possibilities also.

a) steric hindrance between S & I in different sites.

b) overlapping sites for S & I.

c) Partial sharing of sites.

d) Conformational change of enzyme with binding of either such that other can not bind.

Noncompetitive: the inhibitor can bind to either E or ES. S & I do not bind to the same sites (pp 335-40)!

Model: (p 341); and (Table 12.2, p 340).

Note that will have two inhibitor binding constants, they may be the same, as in the equation above, or could be different, leading to more complex behavior.

Plots for classic, simple situation (Figure 12.6 & 12.7):

Uncompetitive: the inhibitor binds ONLY to the ES complex.

Model: (p 340); and (Table 12.2, p 340).

For double reciprocal plots get parallel lines! This is not generally found for single substrate enzymes, but is found in multi-substrate systems.

Multi-substrate Enzymes

Look at three common and easily understood types (p 333-5). We will use Cleland Nomenclature.

• Ordered Sequential Bi Bi mechanism (two on; two off); Note: A must bind first, Q is released last.
• Ping Pong Bi Bi (one on, one off; one on, one off); Note: have some sort of modified enzyme intermediate (often covalent intermediate)
• Random Sequential Bi Bi (two on; two off); Note: A or B may bind first, P or Q may be released last.

Two-substrate Enzyme Product Inhibition Patterns (Based on: E. B. Cunningham, Biochemistry: Mechanisms of Metabolism. McGraw-Hill Book Company, New York (1978), and W. Cleland, "Substrate Inhibition: in Contemporary Enzyme Kinetics and Mechanism. (Daniel L. Purich, ed.) Academic Press, New york (1983)) Kinetic Mechanism Variable Substrate Product Type of Inhibition Ordered Sequential Bi Bi A Q Competitive   B Q Noncompetitive   A P Noncompetitive   B P Noncompetitive Random Sequential Bi Bi A Q Noncompetitive   B Q Noncompetitive   A P Noncompetitive   B P Noncompetitive Ping pong Bi Bi A Q Competitive   B Q Noncompetitive   A P Noncompetitive   B P Competitive

Note that in each case we can predict/explain the pattern of inhibition on the basis of the substrate and inhibitor binding to the same "enzyme form." Thus for the Ordered Sequential mechanism only the first substrate and last product bind to the same form, in this case the free enzyme. Similarly for the Ping pong mechanism the first substrate and last product should be competitive as the both bind the free enzyme. In this case we also see a competitive inhibition between the second substrate and the first product, since they both bind to the E-X complex. The Random Sequential mechanism is a bit more subtle. Here we see across the board noncompetitive since in each case the substrates (and products) can each bind to more than one substrate form, so competitive inhibition will not be possible! (Think of the product as competing with one order of binding but not the other.)

Pathway Diagrams

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Last modified 15 October 2001