IDENTIFICATION OF ADENINE BINDING DOMAIN PEPTIDES OF THE ADP REGULATORY SITE WITHIN GLUTAMATE-DEHYDROGENASE

Photoaffinity labeling with [alpha-P-32]-S-azidoadenosine 5'-diphospha te (8N(3)ADP) and [beta-P-32]-2-azidoadenosine 5'-diphosphate (2N(3)AD P) was used to identify overlapping tryptic and chymotryptic generated peptides within the adenine binding domain of the regulatory ADP site of bovine liver glutamate dehydrogenase (GDH). In the absence of UV i rradiation, 8N(3)ADP was able to activate the reverse reaction catalyz ed by GDH as well as ADP. Photoinsertion of both [alpha(32)P]8N(3)ADP and [beta(32)P]2N(3)ADP was reduced best by ADP in comparison to other nucleotides. Photolabeling of GDH with [alpha(32)P]8N(3)ADP appeared to be biphasic, with saturation occurring near 80 and 130 mu M, wherea s [beta(32)P]2N(3)ADP showed saturation near 50 mu M. When 60 mu M [al pha(32)P]8N(3)ADP (below the first saturation value) was used to ident ify peptides within the ADP binding domain, peptides corresponding to residues G(156)-K-200 and E(175)-K-200 (tryptic) and I-158-Y-183 (chym otryptic) were photolabeled. However, when 160 mu M [alpha(32)P]8N(3)A DP (above the second saturation value) was used, the peptide D-403-R(4 18) was also photolabeled. Digestion with both trypsin and chymotrypsi n resulted in isolation of peptides E(175)-Y-183 and A(184)-I-192 [bet a(32)P]2N(3)ADP at 90 mu M also photolabeled tryptic peptides G(156)-K -200 and C-270-K-299. C-270-K-299 was shown earlier to be within the N AD(+) binding site [Kim, H., and Haley, B. (1991) Bioconjugate Chem. 2 , 142-147]. These results are consistent with the residues E(175)-I-19 2 being within the adenine binding domain of the ADP regulatory site.