title: Glutamic acid gamma-monohydroxamate and hydroxylamine are alternate substrates for Escherichia coli asparagine synthetase B.
abstract: Escherichia coli asparagine synthetase B (AS-B) catalyzes the synthesis of asparagine from aspartic acid and glutamine in an ATP-dependent reaction. The ability of this enzyme to employ hydroxylamine and L-glutamic acid gamma-monohydroxamate (LGH) as alternative substrates in place of ammonia and L-glutamine, respectively, has been investigated. The enzyme is able to function as an amidohydrolase, liberating hydroxylamine from LGH with high catalytic efficiency, as measured by k(cat)/K(M). In addition, the kinetic parameters determined for hydroxylamine in AS-B synthetase activity are very similar to those of ammonia. Nitrogen transfer from LGH to yield aspartic acid beta-monohydroxamate is also catalyzed by AS-B. While such an observation has been made for a few members of the trpG amidotransferase family, our results appear to be the first demonstration that nitrogen transfer can occur from glutamine analogs in a purF amidotransferase. However, k(cat)/K(M) for the ATP-dependent transfer of hydroxylamine from LGH to aspartic acid is reduced 3-fold relative to that for glutamine-dependent asparagine synthesis. Further, the AS-B mutant in which asparagine is replaced by alanine (N74A) can also use hydroxylamine as an alternate substrate to ammonia and catalyze the hydrolysis of LGH. The catalytic efficiencies (k(cat)/K(M)) of nitrogen transfer from LGH and L-glutamine to beta-aspartyl-AMP are almost identical for the N74A AS-B mutant. These observations support the proposal that Asn-74 plays a role in catalyzing glutamine-dependent nitrogen transfer. We interpret our kinetic data as further evidence against ammonia-mediated nitrogen transfer from glutamine in the purF amidotransferase AS-B. These results are consistent with two alternate chemical mechanisms that have been proposed for this reaction [Boehlein, S. K., Richards, N. G. J., Walworth, E. S., & Schuster, S. M. (1994) J. Biol. Chem. 269, 26789-26795].
authors.affiliation: Department of Biochemistry and Molecular Biology, College of Medicine, University of Florida, Gainesville, 32610, USA.
authors.forename: S K
authors.initials: SK
authors.lastname: Boehlein
authors.forename: S M
authors.initials: SM
authors.lastname: Schuster
authors.forename: N G
authors.initials: NG
authors.lastname: Richards
mesh_terms: D001219:Aspartate Aminotransferases / Q000037:antagonists & inhibitors; D001217:Aspartate-Ammonia Ligase / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D005971:Glutamates / Q000378:metabolism*; D006877:Hydroxamic Acids / Q000378:metabolism*; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000378:metabolism*; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D008956:Models, Chemical; D010957:Plasmids; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D004791:Enzyme Inhibitors; D005971:Glutamates; D006877:Hydroxamic Acids; D006898:Hydroxylamines; D007527:Isoenzymes; D011994:Recombinant Proteins; C050614:glutamate-gamma-hydroxamic acid; D019811:Hydroxylamine; D001219:Aspartate Aminotransferases; D001217:Aspartate-Ammonia Ligase