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  • Title: Identification of the lysine and tyrosine peptides labeled by 5'-p-fluorosulfonylbenzoyladenosine in the NADH inhibitory site of glutamate dehydrogenase.
    Author: Schmidt JA, Colman RF.
    Journal: J Biol Chem; 1984 Dec 10; 259(23):14515-9. PubMed ID: 6438099.
    Abstract:
    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.
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