This article 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, for example, in the Omnia assays of epidermal growth factor receptor (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.
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