CHAPTER 5 



DETERMINATION OF THE MECHANISMS 

 AND CONSTANTS OF INHIBITION 



Information on the mechanism of an inhibition may be obtained from 

 accurate kinetic data provided these data are subjected to the appropriate 

 analyses. At the same time it is usually possible to determine the constants 

 that quantitatively characterize the inhibition. A study of enzyme inhibi- 

 tion should ideally involve a determination of the important kinetic and 

 equilibrium constants because the mechanism cannot be adequately ex- 

 pressed otherwise. A mechanism of inhibition in an isolated enzyme system 

 cannot be arrived at without data and procedures capable of supplying 

 these constants nor can the mechanism be applied to more complex 

 situations without a quantitative delineation of the reactions involved. It 

 is, for example, not sufficient to state that an inhibition is competitive 

 without providing the appropriate constants, since if the data are not 

 adequate for the evaluation of the constants they are usually not adequate 

 to prove a competitive mechanism. The mere statement that an enzyme 

 is inhibited to a certain degree by a single concentration of an inhibitor 

 — and such reports are very common — may possibly provide some evi- 

 dence of an enzyme group involved but alone indicates nothing of the mech- 

 anism nor of the behavior of the enzyme and inhibitor in the intact cell. 

 The methods for the calculation of inhibition constants are straightforward 

 in isolated enzyme systems and require only the proper data and a critical 

 choice of the procedures used. The basic approach to such determinations 

 was made by Line weaver and Burk (1934) but other procedures have been 

 developed which are more accurate and applicable in specific cases. True 

 inhibition constants cannot usually be obtained directly from the simple 

 plotting techniques because the constants derived are frequently not the 

 dissociation constants of the enzyme-inhibitor complex but, as is the case 

 with the Michaelis constant, contain other constants or depend on the con- 

 centrations of activators or coenzymes. However, it is generally possible 

 to determine the true inhibition constant if the components of the enzyme 

 are known, if their effects on the inhibition are investigated, and if these 

 data are subjected to the proper graphical methods. It is equally easy to 

 determine constants and to misinterpret them. If inhibition constants are 

 to be useful in any way — whether applied to more complex cellular sys- 



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