Identification of amino acids modified by the bifunctional affinity label 5'-(p-(fluorosulfonyl)benzoyl)-8-azidoadenosine in the reduced coenzyme regulatory site of bovine liver glutamate dehydrogenase.
Bovine liver glutamate dehydrogenase reacts with the bifunctional affinity label 5'-(p-(fluorosulfonyl)benzoyl)-8-azidoadenosine (5'-FSBAzA) in a two-step process: a dark reaction yielding about 0.5 mol of -SBAzA/mol of subunit by reaction through the fluorosulfonyl moiety, followed by photoactivation of the azido group whereby covalently bound -SBAzA becomes cross-linked to the enzyme [Dombrowski, K. E., & Colman, R. F. (1989) Arch. Biochem. Biophys. 275, 302-308]. We now report that the rate constant for the dark reaction is not reduced by ADP or GTP, but it is decreased 7-fold by 2 mM NADH and 40-fold by 2 mM NADH + 0.2 mM GTP, suggesting that 5'-FSBAzA reacts at the GTP-dependent NADH inhibitory site. The amino acid residues modified in each phase of the reaction have been identified. Modified enzyme was isolated after each reaction phase, carboxymethylated, and digested with trypsin, chymotrypsin, or thermolysin. The digests were fractionated by chromatography on a phenylboronate agarose column followed by HPLC. Gas-phase sequencing of the labeled peptides identified Tyr190 as the major amino acid which reacts with the fluorosulfonyl group; Lys143 was also modified but to a lesser extent. The predominant cross-link formed during photolysis is between modified Tyr190 and the peptide Leu475-Asp476-Leu477-Arg478, which is located near the C-terminus of the enzyme. Thus, 5'-FSBAzA is effective in identifying critical residues distant in the linear sequence, but close within the regulatory nucleotide site of glutamate dehydrogenase.
Dombrowski
K E
KE
Department of Chemistry and Biochemistry, University of Delaware, Newark 19716.
Huang
Y C
YC
Colman
R F
RF
D000241:Adenosine / Q000031:analogs & derivatives* / Q000737:chemistry; D000345:Affinity Labels; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism*; D000818:Animals; D001386:Azides / Q000737:chemistry*; D002417:Cattle; D002850:Chromatography, Gel; D003067:Coenzymes / Q000378:metabolism; D003432:Cross-Linking Reagents; D005969:Glutamate Dehydrogenase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism; D010084:Oxidation-Reduction; D010777:Photochemistry
D000345:Affinity Labels; D000596:Amino Acids; D001386:Azides; D003067:Coenzymes; D003432:Cross-Linking Reagents; D009243:NAD; C061559:5'-4-(fluorosulfonyl)benzoyl-8-azidoadenosine; D005969:Glutamate Dehydrogenase; D000241:Adenosine