The –SH Protection Method for Determining Accurate Kd Values for Enzyme-Coenzyme Complexes of NAD+-Dependent Glutamate Dehydrogenase and Engineered Mutants: Evidence for Nonproductive NADPH Complexes

Abstract

Inactivation rates have been measured for clostridial glutamate dehydrogenase and several engineered mutants at various DTNB concentrations. Analysis of rate constants allowed determination of Kd for each non-covalent enzyme-DTNB complex and the rate constant for reaction to form the inactive enzyme-thionitrobenzoate adduct. Both parameters are sensitive to the mutations F238S, P262S, the double mutation F238S/P262S, and D263K, all in the coenzyme binding site. Study of the effects of NAD+, NADH and NADPH at various concentrations in protecting against inactivation by 200 μM DTNB allowed determination of Kd values for binding of these coenzymes to each protein, yielding surprising results. The mutations were originally devised to lessen discrimination against the disfavoured coenzyme NADP(H), and activity measurements showed this was achieved. However, the Kd determinations indicated that, although Kd values for NAD+ and NADH were increased considerably, Kd for NADPH was increased even more than for NADH, so that discrimination against binding of NADPH was not decreased. This apparent contradiction can only be explained if NADPH has a nonproductive binding mode that is not weakened by the mutations, and a catalytically productive mode that, though strengthened, is masked by the nonproductive binding. Awareness of the latter is important in planning further mutagenesis.