Inhibition of glutamate dehydrogenase by covalent coenzyme-substrate adducts: a re-examination.

Covalent adducts of NAD+ with pyruvate and 2-oxoglutarate have been reported to inhibit differentially the activities of bovine glutamate dehydrogenase (GDH) towards these two oxoacid substrates, implying separate active sites. Thorough reinvestigation fails to confirm this finding, with the pyruvate adduct uniformly the more potent inhibitor of both substrate activities under several assay conditions. This suggests that bovine GDH provides amino acid dehydrogenation sites of one structural type only. Clostridial GDH, with a strong preference for oxoglutarate over pyruvate as substrate, is also more strongly inhibited by the pyruvate adduct in the oxoglutarate assay. These findings challenge the generality of the view that carbonyl substrates used in forming such adducts confer specificity for the corresponding substrate binding pocket in enzyme active sites.