Use of nonlinear regression to analyze enzyme kinetic data: application to situations of substrate contamination and background subtraction.

In a recent publication, A. Lundin, P. Arner, and J. Hellmér [Anal. Biochem. 177, 125-131 (1989)] describe a method whereby kinetic substrate assays can be performed when the assay mixture includes a significant contaminating levels of substrate. Their method requires various rearrangements of the data, and involves three separate linear regression calculations. We show how the same data may be analyzed directly, and far more simply, by nonlinear regression. Unlike the linear regression method, nonlinear regression allows direct calculation of the actual values for Km, Vmax, and the concentration of contaminating substrate (as well as estimates of their standard errors); the former method gives only apparent values. The nonlinear regression technique is also statistically a more valid means of analysis, as the rearrangements required to give linearized equations will considerably distort the error distribution and render simple unweighted linear regression inappropriate. The ease of incorporating extra parameters into standard equations when nonlinear regression is used is further illustrated by fitting enzyme reaction data which describe a first-order process when a significant nonspecific background is present. For this equation no simple rearranged linear plot is possible, but nonlinear regression is easily applied to determine the kinetic parameters.