High-throughput screening of enzyme inhibitors: simultaneous determination of tight-binding inhibition constants and enzyme concentration.
Active site titration by a reversible tight-binding inhibitor normally depends on prior knowledge of the inhibition constant. Conversely, the determination of tight-binding inhibition constants normally requires prior knowledge of the active enzyme concentration. Often, neither of these quantities is known with sufficient accuracy. This paper describes experimental conditions under which both the enzyme active site concentration and the tight-binding inhibition constant can be determined simultaneously from a single dose-response curve. Representative experimental data are shown for the inhibition of human kallikrein.
Kuzmic
P
P
BioKin, Ltd., 1652 South Grand Avenue, Suite 337, Pullman, Washington 99163, USA. pkuzmic@biokin.com
Elrod
K C
KC
Cregar
L M
LM
Sideris
S
S
Rai
R
R
Janc
J W
JW
D001665:Binding Sites; D020650:Combinatorial Chemistry Techniques; D004305:Dose-Response Relationship, Drug; D004798:Enzymes / Q000032:analysis* / Q000378:metabolism*; D006801:Humans; D007610:Kallikreins / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000097:blood; D007700:Kinetics; D015233:Models, Statistical; D008962:Models, Theoretical; D009010:Monte Carlo Method; D011485:Protein Binding; D012044:Regression Analysis
D004798:Enzymes; D007610:Kallikreins