Mechanistic interpretation of tryptophan fluorescence quenching in the time courses of glutamate dehydrogenase catalyzed reactions.

We have related the ratios of the protein fluorescence quenching and nucleotide absorbance time courses for the glutamate dehydrogenase catalyzed oxidative deamination of L-glutamate to identify the occurrence and sequential location of a previously demonstrated charge-transfer intermediate. Static studies showed the major portion of the fluorescence quenching signal to be due to radiationless singlet energy transfer from tryptophan to reduced coenzyme chromophores and that conformational changes contribute little to this signal. The ratio approach applied to the transient time courses shows correspondingly that, over most of the time range, the fluorescence quenching signal provides a quantitative measure of the sum of all posthydride transfer species. However, it also indicates the very early occurrence of a species of anomalous optical properties for the reaction catalyzed by the Clostridium symbiosum enzyme as well as that from bovine liver. Transient-state kinetic isotope effect time courses of both the fluorescence and the absorbance signals confirm that this species must be the prehydride charge-transfer complex in both enzyme reactions. Kinetic analysis of alpha-deuterio- and alpha-protio-L-glutamate reaction time courses proves the kinetic competence of the assignments. These results also demonstrate that the intramolecular transfer of a proton from the alpha-amino group of the substrate to an immediately adjacent aspartate carboxylate group on the enzyme is an obligatory initial event in the reactions catalyzed by both enzyme species, even though the occurrence of protein release from a critical lysine residue to the solvent occurs at different phases in those two reactions. The abnormally low intrinsic KIE required to simulate both the alpha-deuterio-L-glutamate reaction and its protio counterpart implies that the transition state of the hydride transfer step must be highly asymmetric.