Initial formation of a non-covalent enzyme-reagent complex during the inactivation of clostridial glutamate dehydrogenase by Ellman's reagent: determination of the enzyme's dissociation constant for the binary complex with NAD+ from protection studies

Abstract

The time-course of reaction between Ellman's reagent (DTNB) and clostridial glutamate dehydrogenase has been investigated over a wide range of reagent concentrations (50–5000μM) and showed pseudo-first-order kinetics throughout. The reaction was followed both by monitoring loss of enzyme activity and by detection of released thionitrobenzoate through its absorbance at 412 nm, and, when both methods were used for the same DTNB concentration, the pseudo-first-order rate constants were identical within experimental error, suggesting that the two methods detect the same process. The dependence of the rate constants on DTNB concentration clearly shows saturation, with a limiting value of 1.62·10-3 s-1 and a dissociation constant of 1.0 mM governing the formation of the implied non-covalent enzyme-DTNB complex. This information has allowed a detailed analysis of the protection of the enzyme by NAD+, yielding a value of 334 μM for the dissociation constant for the enzyme-coenzyme binary complex. In view of the convenience of protection studies as a means of determining dissociation constants, this study emphasizes the importance of establishing whether a chemical modification reaction follows simple first-order kinetics with respect to the chemical reagent.