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Updated: Jul 12, 2026

Steady-state, Pre-steady-state, and Single-turnover Kinetic Measurement for DNA Glycosylase Activity
Published on: August 19, 2013
A theoretical validation of the substrate depletion approach to determining kinetic parameters
Abhinav Nath1, William M Atkins
1Department of Medicinal Chemistry, Box 357610, University of Washington, Seattle, WA 91895-7610, USA.
Abstract:
The substrate depletion approach is an increasingly popular alternative to the traditional method (observing product formation) of determining the kinetic parameters (KM and Vmax) of an enzyme. Obach and Reed-Hagen (Drug Metab Dispos 30:831-837, 2002) used an empirical relationship between substrate depletion rate constants and initial substrate concentration to determine kinetic parameters for a number of cytochrome P450-catalyzed reactions. We present a proof that this relationship can be derived from the Michaelis-Menten equation and, therefore, that kinetic parameters obtained by the substrate depletion approach are equivalent and comparable to those obtained by the traditional product formation approach. Analysis of a simulated data set produced similar kinetic parameters regardless of which approach was used, confirming the theoretical result.
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