Biochemical Pharmacology, Vol. 30. pp. l-7. @ zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Pergamon Press Ltd. 1981. Printed in Great Britain 0006.2952/81/0101-0001 $05.00/O zyxwvut COMMENTARY MEANINGS OF K, FOR CONVENTIONAL AND ALTERNATE- SUBSTRATE INHIBITORS THOMAS SPECTOR*? and W. WALLACE CLELAND$ *Wellcome Research Laboratories, Research Triangle Park, NC 27709, U.S.A., and $Department of Biochemistry, University of Wisconsin, Madison, WI 53706, U.S.A. Inhibition constants (K,) are commonly reported to express the ability of a competitive inhibitor to decrease the velocity of an enzyme-catalyzed reac- tion. Unfortunately, there are frequent misinterpre- tations of the meanings of these constants. For example, the K, value has been considered to be equal to the concentration of inhibitor (I) that pro- duces 50 per cent inhibition of the reaction velocity. This is obviously incorrect because the per cent inhibition will vary with the concentration of the competing substrate. The zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA K, value has also been interpreted to be the dissociation constant (K,) of the inhibitor from the EI complex. This is correct in only a few cases. The relationship between K, and K, can depend upon: (1) the reaction mechanism of the enzyme (i.e. the binding sequence for the sub- t Author to whom all correspondence should be addressed. strates) (2) which substrate is being varied (3) the concentration(s) of the non-varied substrate(s). and (4) whether the inhibitor possesses any alternative substrate activity. The following commentary provides the rationale and simple equations required to convert the variable constant, K,, into the true constant, K,. It also points out how studies with a competitive inhibitor can reveal reaction mechanisms, and explains how the K, for an alternative substrate can be determined from simple competitive inhibition studies. Some of the equations and rules presented here also appear in Refs. l-4. Inhibitors devoid of substrate activity (dead-end inhibitors) Determination of Ki values. Kinetic data are usu- ally presented as double reciprocal plots of the velocity (v) versus the concentration of substrate (S) in the absence and presence of inhibitor (primary PRIMARY PLOT SECONDARY PLOTS SP 30.1. n Fig. 1. Graphical presentations of kinetic data. 1