INITIAL FORMATION OF A NONCOVALENT ENZYME REAGENT COMPLEX DURING THE INACTIVATION OF CLOSTRIDIAL GLUTAMATE-DEHYDROGENASE BY ELLMANS REAGENT- DETERMINATION OF THE ENZYMES DISSOCIATION-CONSTANT FOR THE BINARY COMPLEX WITH NAD(+) FROM PROTECTION STUDIES

The time-course of reaction between Ellman's reagent (DTNB) and clostr idial glutamate dehydrogenase has been investigated over a wide range of reagent concentrations (50-5000 mu M) and showed pseudo-first-order kinetics throughout. The reaction was followed both by monitoring los s of enzyme activity and by detection of released thionitrobenzoate th rough its absorbance at 412 nm, and, when both methods were used for t he 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 format ion 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 mu M for the dissociation constant for th e enzyme-coenzyme binary complex. In view of the convenience of protec tion studies as a means of determining dissociation constants, this st udy emphasizes the importance of establishing whether a chemical modif ication reaction follows simple first-order kinetics with respect to t he chemical reagent.