Reactive amino acid residues involved in glutamate-binding of human glutamate dehydrogenase isozymes.

In the present study, the cassette mutagenesis at several putative positions (K94, G96, K118, K130, or D172) was performed to examine the residues involved in the glutamate-binding of the human glutamate dehydrogenase isozymes (hGDH1 and hGDH2). None of the mutations tested affected the expression or stability of the proteins. There was dramatic reduction in the catalytic efficiency in mutant proteins at K94, G96, K118, or K130 site, but not at D172 site. The K(M) values for glutamate were 4-10-fold greater for the mutants at K94, G96, or K118 site than for the wild-type hGDH1 and hGDH2, whereas no differences in the K(M) values for NAD(+) were detected between the mutant and wild-type enzymes. For K130Y mutant, the K(M) value for glutamate increased 1.6-fold, whereas the catalytic efficiency (k(cat)/K(M)) showed only 2-3% of the wild-type. Therefore, the decreased catalytic efficiency of the K130 mutant mainly results from the reduced k(cat) value, suggesting a possibility that the K130Y residue may be involved in the catalysis rather than in the glutamate-binding. The D172Y mutant did not show any changes in k(cat) value and K(M) values for glutamate and NAD(+), indicating that D172Y is not directly involved in catalysis and substrates binding of the hGDH isozymes. For sensitivity to ADP activation, only the D172Y mutant showed a reduced sensitivity to ADP activation. The reduction of ADP activation in D172Y mutant was more profoundly observed in hGDH2 than in hGDH1. There were no differences in their sensitivities to GTP inhibition between the wild-type and mutant GDHs at all positions tested. Our results suggest that K94, G96, and K118 residues play an important role, although at different degrees, in the binding of glutamate to hGDH isozymes.