Boehlein S K, Schuster S M, Richards N G
Department of Biochemistry and Molecular Biology, College of Medicine, University of Florida, Gainesville, 32610, USA.
Biochemistry. 1996 Mar 5;35(9):3031-7. doi: 10.1021/bi952505l.
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].
大肠杆菌天冬酰胺合成酶B（AS-B）在依赖ATP的反应中催化由天冬氨酸和谷氨酰胺合成天冬酰胺。该酶分别利用羟胺和L-谷氨酸γ-单羟肟酸酯（LGH）替代氨和L-谷氨酰胺作为替代底物的能力已得到研究。通过k(cat)/K(M)测定，该酶能够作为酰胺水解酶发挥作用，以高催化效率从LGH中释放羟胺。此外，为AS-B合成酶活性中的羟胺测定的动力学参数与氨的动力学参数非常相似。AS-B还催化从LGH到天冬氨酸β-单羟肟酸酯的氮转移。虽然对trpG酰胺转移酶家族的一些成员有过这样的观察，但我们的结果似乎是首次证明在purF酰胺转移酶中可以从谷氨酰胺类似物发生氮转移。然而，相对于依赖谷氨酰胺的天冬酰胺合成，从LGH到天冬氨酸的依赖ATP的羟胺转移的k(cat)/K(M)降低了3倍。此外，天冬酰胺被丙氨酸取代的AS-B突变体（N74A）也可以使用羟胺作为氨的替代底物并催化LGH的水解。对于N74A AS-B突变体，从LGH和L-谷氨酰胺到β-天冬氨酰-AMP的氮转移的催化效率（k(cat)/K(M)）几乎相同。这些观察结果支持Asn-74在催化依赖谷氨酰胺的氮转移中起作用的提议。我们将动力学数据解释为反对在purF酰胺转移酶AS-B中氨介导的从谷氨酰胺的氮转移的进一步证据。这些结果与针对该反应提出的两种替代化学机制一致[Boehlein, S. K., Richards, N. G. J., Walworth, E. S., & Schuster, S. M. (1994) J. Biol. Chem. 269, 26789 - 26795]。
