GLUCOSAMINE SYNTHETASE FROM ESCHERICHIA-COLI - PURIFICATION, PROPERTIES, AND GLUTAMINE-UTILIZING SITE LOCATION
GLUCOSAMINE SYNTHETASE FROM ESCHERICHIA-COLI - PURIFICATION, PROPERTIES, AND GLUTAMINE-UTILIZING SITE LOCATION
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DOI：
10.1021/bi00381a023
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发表时间：
1987-04-07
期刊：
影响因子：
2.9
通讯作者：
LEGOFFIC, F
中科院分区：
文献类型：
--
作者：
BADET, B;VERMOOTE, P;LEGOFFIC, F
L-谷氨酰胺：D-果糖-6-磷酸酰胺转移酶（葡萄糖胺合成酶）已从大肠杆菌中纯化至同质。通过十二烷基硫酸钠凝胶电泳估计亚基分子量为 70 800。纯的葡萄糖胺合成酶不表现出可检测的 NH3 依赖性活性，并且不催化逆反应，正如对更多不纯制剂所报道的那样 [Gosh, S.、Blumenthal, H. J.、Davidson, E. 和 Roseman, S (1960) J. Biol.化学。 235、1265]。该酶对于 6-磷酸果糖的 Km 为 2 mM，对于谷氨酰胺的 Km 为 0.4 mM，周转数为 1140 min-1。氨基末端序列证实了翻译的大肠杆菌glmS序列的残基2-26的鉴定[Walker，J.E.，Gay，J.，Saraste，M.，和Eberle，N.(1984)Biochem。 J. 224, 799]。因此，甲硫氨酸-1 通过体内加工被去除，留下半胱氨酸作为 NH2 末端残基。该酶被谷氨酰胺类似物 6-重氮-5-氧代-L-正亮氨酸 (DON) 和碘乙酰胺灭活。在不存在 6-磷酸果糖的情况下，与 DON 一起孵育时，葡萄糖胺合成酶表现出一半的位点反应性。在其存在下，[6-14C]DON 灭活伴随着每个酶亚基掺入 1 当量的抑制剂。根据这种行为，二聚体结构被暂时归属于天然酶。通过 Edman 降解显示，与 DON 的反应位点是 NH2 末端半胱氨酸残基。
L-Glutamine:D-fructose-6-phosphate amidotransferase(glucosamine synthetase) has been purified to homogeneity from Escherichia coli. A subunit molecular weight of 70 800 was estimated by gel electrophoresis in sodium dodecyl sulfate. Pure glucosamine synthetase did not exhibit detectable NH3-dependent activity and did not catalyze the reverse reaction, as reported for more impure preparations [Gosh, S., Blumenthal, H. J., Davidson, E., and Roseman, S (1960) J. Biol. Chem. 235, 1265]. The enzyme has a Km of 2 mM for fructose 6-phosphate, a Km of 0.4 mM for glutamine, and a turnover number of 1140 min-1. The amino-terminal sequence confirmed the identification of residues 2-26 of the translated E. coli glmS sequence [Walker, J. E., Gay, J., Saraste, M., and Eberle, N. (1984) Biochem. J. 224, 799]. Methionine-1 is therefore removed by processing in vivo, leaving cysteine as the NH2-terminal residue. The enzyme was inacivated by the glutamine analogue 6-diazo-5-oxo-L-norleucine (DON) and by iodoacetamide. Glucosamine synthetase exhibited half-of-the sites reactivity when incubated with DON in the absence of fructose 6-phosphate. In its presence, inactivation with [6-14C]DON was accompanied by incorporation of 1 equiv of inhibitor per enzyme subunit. From this behavior, a dimeric structure was tentatively assigned to the native enzyme. The site of reaction with DON was the NH2-terminal cysteine residue as shown by Edman degradation.