Analysis of slow-binding enzyme inhibitors at elevated enzyme concentrations

The improvement in the characterization of slow-binding inhibitors achieved by performing experiments at elevated enzyme concentrations is presented. In particular, the characterization of slow-binding inhibitors conforming to a two-step mode of inhibition with a steady-state dissociation constant t...

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Bibliographic Details
Published in:Analytical biochemistry 2005-02, Vol.337 (2), p.211-223
Main Authors: Perdicakis, Basil, Montgomery, Heather J., Guillemette, J. Guy, Jervis, Eric
Format: Article
Language:English
Subjects:
Enzyme assay
Enzyme Inhibitors - analysis
Enzyme Inhibitors - metabolism
Enzymes - metabolism
Humans
Inhibitor
Kinetics
MATLAB
Models, Chemical
Multiwell plate
Nitric oxide
Nitric oxide synthase
Nitric Oxide Synthase - metabolism
Nitric Oxide Synthase Type II
Oxyhemoglobin
Progress curve
Regression
Slow-binding
Time Factors
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Summary:The improvement in the characterization of slow-binding inhibitors achieved by performing experiments at elevated enzyme concentrations is presented. In particular, the characterization of slow-binding inhibitors conforming to a two-step mode of inhibition with a steady-state dissociation constant that is much lower than the initial dissociation constant with enzyme is discussed. For these systems, inhibition is rapid and low steady-state product concentrations are produced at saturating inhibitor concentrations. By working at elevated enzyme concentrations, improved signal-to-noise ratios are achieved and data may be collected at saturating inhibitor levels. Numerical simulations confirmed that improved parameter estimates are obtained and useful data to discern the mechanism of slow-binding inhibition are produced by working at elevated enzyme concentrations. The saturation kinetics that were unobservable in two previous studies of an enzyme inhibitor system were measured by performing experiments at an elevated enzyme concentration. These results indicate that consideration of the quality of the data acquired using a particular assay is an important factor when selecting the enzyme concentration at which to perform experiments used to characterize the class of enzyme inhibitors examined herein.
ISSN:0003-2697
1096-0309
DOI:10.1016/j.ab.2004.11.012