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 i...

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Bibliographic Details
Published in:Analytical biochemistry 2000-11, Vol.286 (1), p.45-50
Main Authors: Kuzmič, Petr, Elrod, Kyle C, Cregar, Lynne M, Sideris, Steve, Rai, Roopa, Janc, James W
Format: Article
Language:English
Subjects:
active site titration
Binding Sites
Combinatorial Chemistry Techniques
Dose-Response Relationship, Drug
enzyme concentration
enzyme kinetics
Enzymes - analysis
Enzymes - metabolism
high-throughput screening
Humans
inhibition constant
kallikrein
Kallikreins - analysis
Kallikreins - antagonists & inhibitors
Kallikreins - blood
Kinetics
mathematics
Models, Statistical
Models, Theoretical
Monte Carlo Method
Protein Binding
Regression Analysis
statistics
tight binding
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Summary: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.
ISSN:0003-2697
1096-0309
DOI:10.1006/abio.2000.4685