title: Adenosine 5'-0-[S-(4-succinimidyl-benzophenone)thiophosphate]: a new photoaffinity label of the allosteric ADP site of bovine liver glutamate dehydrogenase.
abstract: By reaction of adenosine 5'-monothiophosphate with benzophenone-4-maleimide, we synthesized adenosine 5'-O-[S-(4-succinimidyl-benzophenone)thiophosphate] (AMPS-Succ-BP) as a photoreactive ADP analogue. Bovine liver glutamate dehydrogenase is known to be allosterically activated by ADP, but the ADP site has not been located in the crystal structure of the hexameric enzyme [Peterson, P. E., and Smith, T. J. (1999) Structure 7, 769-782]. In the dark, AMPS-Succ-BP reversibly activates GDH. Irradiation of the complex of glutamate dehydrogenase and AMPS-Succ-BP at lambda >300 nm causes a time-dependent, irreversible 2-fold activation of the enzyme. The k(obs) for photoactivation shows nonlinear dependence on the concentration of AMPS-Succ-BP, with K(R) = 4.9 microM and k(max) = 0.076 min(-)(1). The k(obs) for photoreaction by 20 microM AMPS-Succ-BP is decreased 10-fold by 200 microM ADP, but is reduced less than 2-fold by NAD, NADH, GTP, or alpha-ketoglutarate. Modified enzyme is no longer activated by ADP, but is still inhibited by GTP and high concentrations of NADH. These results indicate that reaction of AMPS-Succ-BP occurs within the ADP site. The enzyme incorporates up to 0.5 mol of [(3)H]AMPS-Succ-BP/mol of enzyme subunit or 3 mol of reagent/mol of hexamer. The peptide Lys(488)-Glu(495) has been identified as the only reaction target, and the data suggest that Arg(491) is the modified amino acid. Arg(491) (in the C-terminal helix close to the GTP #2 binding domain of GDH) is thus considered to be at or near the enzyme's allosteric ADP site. On the basis of these results, the AMPS-Succ-BP was positioned within the crystal structure of glutamate dehydrogenase, where it should also mark the ADP binding site of the enzyme.
authors.affiliation: Department of Chemistry and Biochemistry, University of Delaware, Newark, DE 19716, USA.
authors.forename: K S
authors.initials: KS
authors.lastname: Madhusoodanan
authors.forename: R F
authors.initials: RF
authors.lastname: Colman
mesh_terms: D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism*; D000495:Allosteric Site; D000818:Animals; D001577:Benzophenones / Q000737:chemistry / Q000378:metabolism*; D002417:Cattle; D004789:Enzyme Activation; D005969:Glutamate Dehydrogenase / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D008099:Liver / Q000201:enzymology*; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism; D019920:Photoaffinity Labels / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D013211:Staphylococcus aureus / Q000201:enzymology; D013388:Succinimides / Q000737:chemistry / Q000378:metabolism*; D013873:Thionucleotides / Q000378:metabolism*; D014316:Tritium
chemical_list: D001577:Benzophenones; D008024:Ligands; D010446:Peptide Fragments; D019920:Photoaffinity Labels; D013388:Succinimides; D013873:Thionucleotides; C426043:adenosine 5'-0-(S-(4-succinimidyl-benzophenone)thiophosphate); D014316:Tritium; D000244:Adenosine Diphosphate; D005969:Glutamate Dehydrogenase; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase