Ultraviolet Spectrophotometric Characterization of a Glutamate Dehydrogenase-reduced Coenzyme-α-Ketoglutarate Complex

Glutamate dehydrogenase binds α-ketoglutarate and TPNH in a ternary complex such that both ligands are more tightly bound than in binary complexes. TPNH, bound in this ternary complex, absorbs maximally at 332 nm with E332 = 5.2 mm-1 cm-1 in contrast to the absorbance of free TPNH (E339 = Emax = 6.2 mm-1 cm-1) and TPNH bound in either a binary or in a glutamate dehydrogenase-TPNH-l-glutamate dead-end ternary complex (E348 = Emax = 4.8 mm-1 cm-1). One molecule of TPNH per 47,000 to 54,000 molecular weight glutamate dehydrogenase (approximately one per peptide chain) was found to be bound in either the α-ketoglutarate or l-glutamate ternary complex. The spectral shifts associated with ternary complex formation were not observed when the coenzyme and substrate analogs, 3-acetylpyridine DPNH or α-ketobutyrate, were used. Therefore, an intact amide on the reduced nicotinamide moiety and a gamma-carboxyl of the dicarboxylate substrate must be required for observable complex formation. The demonstration of the high stability of the glutamate dehydrogenase-TPNH-α-ketoglutarate complex and the correlation of the binding characteristics of the ligands involved in this complex with the effects of these ligands on catalytic events warrants the inclusion of this complex as either a reactive intermediate or a dead-end complex in all general mechanisms proposed for glutamate dehydrogenase catalysis.