Importance of glutamate 279 for the coenzyme binding of human glutamate dehydrogenase.

Abstract Although the structure of glutamate dehydrogenase (GDH) has been reported from various sources including mammalian GDH, there are conflicting views regarding the location and mechanism of actions of the coenzyme binding. We have expanded these speculations by photoaffinity labeling and cassette mutagenesis. Photoaffinity labeling with a specific probe, [32P]nicotinamide 2-azidoadenosine dinucleotide, was used to identify the NAD+ binding site within human GDH encoded by the synthetic human GDH gene and expressed inEscherichia coli as a soluble protein. Photolabel-containing peptides generated with trypsin were isolated by immobilized boronate affinity chromatography. Photolabeling of these peptides was most effectively prevented by the presence of NAD+ during photolysis, demonstrating a selectivity of the photoprobe for the NAD+ binding site. Amino acid sequencing and compositional analysis identified Glu279 as the site of photoinsertion into human GDH, suggesting that Glu279 is located at or near the NAD+ binding site. The importance of the Glu279 residue in the binding of NAD+ was further examined by cassette mutagenesis with mutant enzymes containing Arg, Gly, Leu, Met, or Tyr at position 279. The mutagenesis at Glu279 has no effects on the expression or stability of the different mutants. The K mvalues for NAD+ were 10–14-fold greater for the mutant GDHs than for wild-type GDH, whereas the V maxvalues were similar for wild-type and mutant GDHs. The efficiency (k cat/K m) of the mutant GDH was reduced up to 18-fold. The decreased efficiency of the mutants results from the increase in K m values for NAD+. In contrast to the K m values for NAD+, wild-type and mutant GDHs show similarK m values for glutamate, indicating that substitution at position 279 had no appreciable effect on the affinity of enzyme for glutamate. There were no differences in sensitivities to ADP activation and GTP inhibition between wild-type and mutant GDH, suggesting that Glu279 is not directly involved in allosteric regulation. The results with photoaffinity labeling and cassette mutagenesis studies suggest that Glu279 plays an important role for efficient binding of NAD+ to human GDH.