Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.4.1.2 (glutamate dehydrogenase)
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The fluorescent nucleotide analogue 5'-[p-(fluorosulfonyl)benzoyl]-1,N6-ethenoadenosine (5'-FSB epsilon A) reacts irreversibly with bovine liver glutamate dehydrogenase and modifies one of the natural inhibitory guanosine 5'-triphosphate (GTP) sites [Jacobson, M.A., & Colman, R.F. (1982) Biochemistry 21, 2177-2186]. Enzyme with 1.28 mol of 5'-(p-sulfonylbenzoyl)-1,N6-ethenoadenosine/mol of subunit incorporated and exhibiting maximum change in sensitivity to GTP inhibition is now shown by amino acid analysis to contain 0.95 mol of O-[(4-carboxyphenyl)sulfonyl]tyrosine (CBS-Tyr) and 0.33 mol of N epsilon-[(4-carboxyphenyl)sulfonyl]-lysine (CBS-Lys), quantitatively accounting for the total incorporation prior to acid hydrolysis. As a function of time of incubation with 5'-FSB epsilon A, the amount of CBS-Tyr formed was directly proportional to the change in GTP inhibition. In contrast, an initial formation of CBS-Lys was observed, followed by relatively little additional CBS-Lys although the percent change in GTP inhibition continued to increase. It was concluded that the tyrosine is an essential residue in the GTP binding site of glutamate dehydrogenase, while the lysine modified is not involved in the inhibitory action of GTP. The nucleotide analogue 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-diphosphate (TNP-ADP) was evaluated for its ability to occupy the adenosine 5'-diphosphate (ADP) activator site and to function as an energy acceptor conjointly with 5'-SB epsilon A covalently bound at the GTP site as the energy donor. TNP-ADP activates native enzyme 2-fold and competes kinetically with ADP. As determined by fluorometric titration, the maximum number of TNP-ADP binding sites on native enzyme was 0.5 mol/mol of subunit in the absence and 1 mol/mol of subunit in the presence of reduced coenzyme. The 5'-SB epsilon A-modified enzyme also binds TNP-ADP: 0.5 mol/mol of subunit in the absence or presence of reduced coenzyme. TNP-ADP competes for binding with ADP to native and 5'-SB epsilon A-modified enzyme, indicating that this nucleotide analogue is a satisfactory fluorescent probe of the ADP site of glutamate dehydrogenase. An energy-transfer efficiency of 0.77 was determined from the decrease in donor fluorescence upon addition of TNP-ADP in the absence of reduced coenzyme to modified enzyme containing 1.23 mol of 5'-SB epsilon A/mol of subunit. A value of 18 A was calculated as the average distance between the GTP and ADP regulatory sites. This result indicates that the inhibitory GTP and the activatory ADP sites are close but not identical.
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PMID:Resonance energy transfer between the adenosine 5'-diphosphate site of glutamate dehydrogenase and a guanosine 5'-triphosphate site containing a tyrosine labeled with 5'-[p-(fluorosulfonyl)benzoyl]-1,N6-ethenoadenosine. 641 7

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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PMID:Identification of the lysine and tyrosine peptides labeled by 5'-p-fluorosulfonylbenzoyladenosine in the NADH inhibitory site of glutamate dehydrogenase. 643 99