GLUTAMIC-ACID GAMMA-MONOHYDROXAMATE AND HYDROXYLAMINE ARE ALTERNATE SUBSTRATES FOR ESCHERICHIA-COLI ASPARAGINE SYNTHETASE-B

Escherichia coli asparagine synthetase B (AS-B) catalyzes the synthesi s of asparagine from aspartic acid and glutamine in an ATP-dependent r eaction. The ability of this enzyme to employ hydroxylamine and L-glut amic acid gamma-monohydroxamate (LGH) as alternative substrates in pla ce of ammonia and L-glutamine, respectively, has been investigated. Th e enzyme is able to function as an amidohydrolase, liberating hydroxyl amine 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. Nitr ogen 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 glut amine analogs in a purF amidotransferase. However, k(cat)/K-M for the ATP-dependent transfer of hydroxylamine from LGH to aspartic acid is r educed 3-fold relative to that for glutamine-dependent asparagine synt hesis. Further, the AS-B mutant in which asparagine is replaced by ala nine (N74A) can also use hydroxylamine as an alternate substrate to am monia and catalyze the hydrolysis of LGH. The catalytic efficiencies ( k(cat)/K-M) of nitrogen transfer from LGH and L-glutamine to beta-aspa rtyl-AMP are almost identical for the N74A AS-B mutant. These observat ions support the proposal that Asn-74 plays a role in catalyzing gluta mine-dependent nitrogen transfer. We interpret our kinetic data as fur ther evidence against ammonia-mediated nitrogen transfer from glutamin e in the purF amidotransferase AS-B, These results are consistent with two alternate chemical mechanisms that have been proposed for this re action [Boehlein, S. K., Richards, N. G., J., Walworth, E. S., & Schus ter, S. M. (1994) J. Biol. Chem, 269, 26789-26795].