Photoaffinity labeling with [gamma-32P]8N3GTP (8-azidoguanosine triphosphate) was used to identify the guanine binding peptides of the GTT binding site within two types of glutamate dehydrogenase isoproteins (GDH I and GDH II) isolated from bovine brain. 8N3GTP, without photolysis, mimicked the inhibitory properties of GTP on GDH I and GDH II activities. Saturation of photoinsertion of GDH isoproteins revealed an apparent Kd of 8 microM (GDH I) and 24 microM (GDH II) for [gamma-32P]8N3GTP. Ion exchange and reversed-phase high-performance liquid chromatography (HPLC) were used to isolate photolabel-containing peptides generated with trypsin. This identified a portion of the guanine binding domain within the GTP binding site is the region containing the sequence I-S-G-A-S-E-X-D-I-V-H-S-A-L-A-Y-T-M E-R (GDH I) and I-S-G-A-S-E-X-D-I-V-H-S-G-L-A-Y-T-M-E-R (GDH II). The symbol X indicates a position for which no phenylthiohydantoin-amino acid could be assigned. The missing residue, however, can be designated as a photolabeled lysine since the sequences including the lysine residue in question have a complete identity with those of the other GDH species known. Also, trypsin was unable to cleave the photolabeled peptide at this site. Photolabeling of these peptides was prevented by the presence of GTP during photolysis, while other nucleotides could not reduce the amount of photoinsertion as effectively as GTP. These results demonstrate selectivity of the photoprobe for the GTP binding site and suggest that the peptide identified using the photoprobe is located in the GTP binding domain of the brain GDH isoproteins.