Identification of adenine binding domain peptides of the ADP regulatory site within glutamate dehydrogenase.
Photoaffinity labeling with [alpha-32P]-8-azidoadenosine 5'-diphosphate (8N3ADP) and [beta-32P]-2-azidoadenosine 5'-diphosphate (2N3ADP) 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 irradiation, 8N3ADP was able to activate the reverse reaction catalyzed by GDH as well as ADP. Photoinsertion of both [alpha 32P] 8N3ADP and [beta 32P]2N3ADP was reduced best by ADP in comparison to other nucleotides. Photolabeling of GDH with [alpha 32P]8N3-ADP appeared to be biphasic, with saturation occurring near 80 and 130 microM, whereas [beta 32P]2N3ADP showed saturation near 50 microM. When 60 microM [alpha 32P]8N3ADP (below the first saturation value) was used to identify peptides within the ADP binding domain, peptides corresponding to residues G156-K200 and E175-K200 (tryptic) and I158-Y183 (chymotryptic) were photolabeled. However, when 160 microM [alpha 32P]8N3ADP (above the second saturation value) was used, the peptide D403-R418 was also photolabeled. Digestion with both trypsin and chymotrypsin resulted in isolation of peptides E175-Y183 and A184-I192. [beta 32P]2N3ADP at 90 microM also photolabeled tryptic peptides G156-K200 and C270-K289. C270-K289 was shown earlier to be within the NAD+ binding site [Kim, H., and Haley, B. (1991) Bioconjugate Chem. 2, 142-147]. These results are consistent with the residues E175-[192 being within the adenine binding domain of the ADP regulatory site.
Shoemaker
M T
MT
Department of Biochemistry, College of Medicine, College of Pharmacy, University of Kentucky Medical Center, Lexington.
Haley
B E
BE
D000225:Adenine / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism*; D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D001386:Azides / Q000737:chemistry; D001665:Binding Sites; D002417:Cattle; D002847:Chromatography, Agarose / Q000379:methods; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002918:Chymotrypsin / Q000378:metabolism; D004853:Epoxy Resins / Q000737:chemistry; D005969:Glutamate Dehydrogenase / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism*; D008099:Liver / Q000737:chemistry; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000737:chemistry / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D017421:Sequence Analysis; D013053:Spectrophotometry; D014357:Trypsin / Q000378:metabolism
D000345:Affinity Labels; D001386:Azides; D004853:Epoxy Resins; D009711:Nucleotides; D010761:Phosphorus Radioisotopes; D000244:Adenosine Diphosphate; C022295:2-azidoadenosine 3',5'-diphosphate; D005969:Glutamate Dehydrogenase; D002918:Chymotrypsin; D014357:Trypsin; C021622:8-azidoadenosine diphosphate; D000225:Adenine