This report describes an integrated rate equation for the time-course of covalent enzyme
inhibition under the conditions where the substrate concentration is significantly lower
than the corresponding Michaelis constant, such as for example in the Omnia assays
of EGFR kinase. The newly described method is applicable to experimental conditions
where the enzyme concentration is significantly lower than the dissociation constant of
the initially formed reversible enzyme-inhibitor complex (no "tight binding"). A detailed
comparison with the traditionally used rate equation for covalent inhibition is presented.
The two methods produce approximately identical values of the first-order inactivation
rate constant (kinact). However, the inhibition constant (Ki) and therefore also the
second-order inactivation rate constant kinact/Ki, is underestimated by the traditional
method by up to an order of magnitude.
enzyme kinetics; mathematics
How to Cite
Kuzmic, P. (2015) Determination of substrate kinetic parameters from progress curve data,
Anal. Biochem., In Press [Manuscript No. ABIO-14-632R1]
This Technical Note is the precursor form of an article to be published later in 2015 in the journal Analytical Biochemistry.
The current text and illustrations are exactly as originally submitted to the Journal, prior to any editorial changes or
other processing performed by the Publisher.
www.biokin.com/publications/technotes/TN201504.html Tue Nov 29 06:58:35 2016