Identification of a guanine binding domain peptide of the GTP binding site of glutamate dehydrogenase: isolation with metal-chelate affinity chromatography.
Photoaffinity labeling with [alpha-32P]8N3GTP and [gamma-32P]8N3GTP was used to identify the guanine binding domain of the GTP regulatory site within glutamate dehydrogenase (GDH). Without photolysis, 8N3GTP mimicked the regulatory properties of GTP on GDH activity with 8N3GTP exhibiting a Ki of 5 microM while the Ki for GTP was about 0.6 microM. Under optimal photolabeling conditions saturation of photoinsertion with 1 microgram of GDH revealed an apparent Kd of 9 +/- 4 microM for [gamma-32P]8N3GTP. Photolabeling with this analog could be competitively inhibited with GTP with an apparent Kd of 12 +/- 2 microM. Other nucleotides such as ATP and NAD(P)H could not reduce the amount of photoinsertion as effectively as GTP. ADP could decrease photoinsertion, but only at much higher concentrations. NAD(P)+, GDP, AMP, and GMP had little effect on photoinsertion. Divalent cations Mg2+ and Ca2+ also reduced photoinsertion significantly while the monovalent K+ and Na+ ions had no effect. Aluminum(III)-chelate or iron(III)-chelate affinity chromatography and reversed-phase HPLC were used to purify photolabel-containing peptides generated with either trypsin or chymotrypsin. This identified a portion of the guanine binding domain within the GTP regulatory site as the region containing the sequence Ile439 to Tyr454. Photolabeling of this peptide was prevented 91% by the presence of 300 microM GTP during photolysis. Lys445 was not identified in sequence analyses of the photolabeled peptides. Also, trypsin was unable to cleave the photolabeled peptide at this site. These results suggest that Lys445 may be the residue modified by [alpha-32P]8N3GTP.
Shoemaker
M T
MT
Division of Medicinal Chemistry & Pharmaceutics, College of Pharmacy, University of Kentucky Medical Center, Lexington 40536-0093.
Haley
B E
BE
D000345:Affinity Labels; D000535:Aluminum; D000595:Amino Acid Sequence; D000818:Animals; D001386:Azides / Q000378:metabolism / Q000494:pharmacology; D001665:Binding Sites; D001667:Binding, Competitive; D002118:Calcium / Q000494:pharmacology; D002413:Cations, Divalent; D002417:Cattle; D002614:Chelating Agents; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D005969:Glutamate Dehydrogenase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D006147:Guanine / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D007502:Iron Chelating Agents; D008027:Light; D008274:Magnesium / Q000494:pharmacology; D008969:Molecular Sequence Data; D010777:Photochemistry
C021961:8-azidoguanosine triphosphate; D000345:Affinity Labels; D001386:Azides; D002413:Cations, Divalent; D002614:Chelating Agents; D007502:Iron Chelating Agents; D006147:Guanine; D006160:Guanosine Triphosphate; D000535:Aluminum; D005969:Glutamate Dehydrogenase; D008274:Magnesium; D002118:Calcium