Implication of arginine-131 and arginine-303 in the substrate site of adenylosuccinate synthetase of Escherichia coli by affinity labeling with 6-(4-bromo-2,3-dioxobutyl)thioadenosine 5'-monophosphate.
Adenylosuccinate synthetase from Escherichia coli is inactivated in a biphasic reaction by 6-(4-bromo-2,3-dioxobutyl)thioadenosine 5'-monophosphate (6-BDB-TAMP) at pH 7.0 and 25 degrees C. The initial fast-phase inactivation is not affected by the presence of active-site ligands and can be completely eliminated by blocking Cys291 of the enzyme with N-ethylmaleimide (NEM). Reaction of the NEM-treated enzyme with 6-BDB-[32P]TAMP results in 2 mol of reagent incorporated/mol of enzyme subunit. The inactivation kinetics of the slow-phase exhibit an apparent KI of 40.6 microM and kmax of 0.0228 min-1. Active-site ligands, either adenylosuccinate or IMP and GTP, completely prevent inactivation of the enzyme by 6-BDB-TAMP, whereas IMP or IMP and aspartate is much less effective in protection. 6-BDB-TAMP-inactivated enzyme has a 3-fold increase in Km for aspartate with no change in Km for IMP or GTP. Protease digestion of 6-BDB-[32P]TAMP inactivated enzyme reveals that both Arg131 and Arg303 are modified by the affinity-labeling reagent. The crystal structure [Poland, B. W., Fromm, H. J., and Honzatko, R. B. (1996) J. Mol. Biol. 264, 1013-1027] and site-directed mutagenesis [Kang, C., Sun, N., Poland, B. W., Gorrell, A., and Fromm, H. J. (1997) J. Biol. Chem. 272, 11881-11885] of E. coli adenylosuccinate synthetase show that Arg303 interacts with the carboxyl group of aspartate and the 2'-OH of the ribose of IMP and Arg131 is involved in stabilizing aspartate in the active site of the enzyme. We conclude that 6-BDB-TAMP functions as a reactive adenylosuccinate analogue in modifying both Arg131 and Arg303 in the active site of adenylosuccinate synthetase.
Lee
P
P
Department of Chemistry and Biochemistry, University of Delaware, Newark 19716, USA.
Gorrell
A
A
Fromm
H J
HJ
Colman
R F
RF
D000249:Adenosine Monophosphate / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D000265:Adenylosuccinate Synthase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D000345:Affinity Labels / Q000378:metabolism* / Q000494:pharmacology; D001120:Arginine / Q000378:metabolism*; D001665:Binding Sites; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli / Q000201:enzymology*; D005033:Ethylmaleimide / Q000494:pharmacology; D007700:Kinetics; D008024:Ligands; D010449:Peptide Mapping; D013379:Substrate Specificity
D000345:Affinity Labels; D008024:Ligands; D000249:Adenosine Monophosphate; C042297:6-(6-(4-bromo-2,3-dioxobutyl)thio)deaminoadenosine 5'-monophosphate; D001120:Arginine; D000265:Adenylosuccinate Synthase; D005033:Ethylmaleimide