Identification of the lysine and tyrosine peptides labeled by 5'-p-fluorosulfonylbenzoyladenosine in the NADH inhibitory site of glutamate dehydrogenase.
5'-p-Fluorosulfonylbenzoyladenosine (5'-FSBA) is a specific affinity label for the inhibitory NADH site of bovine liver glutamate dehydrogenase. Reaction of the enzyme with 5'-FSBA results in the loss of inhibition by high concentrations of NADH with covalent attachment of 0.53 sulfonylbenozyladenosine/subunit, i.e. modification of three subunits of the hexameric enzyme. Equal amounts of N epsilon-(4-carboxybenzenesulfonyl)lysine (Lys-(CBS] and O-(4-carboxybenzenesulfonyl)tyrosine (Tyr-(CBS] are found throughout the course of the reaction (Saradambal, K. V., Bednar, R. A., and Colman, R. F. (1981) J. Biol. Chem. 256, 11866-11872). Modified enzyme, prepared by incubating 2 mg/ml glutamate dehydrogenase with 0.3 mM 3H-labeled 5'-FSBA at pH 8 for 1 h, was carboxymethylated and digested with thermolysin. Two nucleosidyl peptides were isolated by a combination of chromatography on phenyl boronate-agarose, high-performance liquid chromatography in ammonium bicarbonate and high-performance liquid chromatography in trifluoroacetic acid. By comparison of the amino acid analysis and NH2-terminal residue of each isolated peptide with the known amino acid sequence of the enzyme, the peptides were identified as Leu-Gly-Arg-Lys(CBS) and Ile-Gly-His-Tyr(CBS)-Asp. These sequences correspond to residues 417-420 and 187-191, respectively. Lys-420 and Tyr-190 of glutamate dehydrogenase react with 5'-FSBA, and both are apparently located in the NADH inhibitory site.
D000241:Adenosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000345:Affinity Labels / Q000494:pharmacology*; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002417:Cattle; D005969:Glutamate Dehydrogenase / Q000037:antagonists & inhibitors*; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008239:Lysine / Q000032:analysis*; D009243:NAD / Q000494:pharmacology*; D010084:Oxidation-Reduction; D014443:Tyrosine / Q000032:analysis*
D000345:Affinity Labels; D000596:Amino Acids; D009243:NAD; D014443:Tyrosine; C010959:5'-(4-fluorosulfonylbenzoyl)adenosine; D005969:Glutamate Dehydrogenase; D008239:Lysine; D000241:Adenosine