Glutamine-dependent氮转移在大肠杆菌天冬酰胺合成酶寻找催化三分子。

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Boehlein SK,理查兹NG,舒斯特尔SM

Glutamine-dependent氮转移在大肠杆菌天冬酰胺合成酶寻找催化三分子。

J生物化学杂志。1994年3月11日,269 (10):7450 - 7。

PubMed ID

7907328 (在PubMed

]

文摘

氮转移的机制glutamine-dependent amidotransferases有待明确建立。我们现在报告超表达、纯化和动力学特性的大肠杆菌的谷氨酰胺和ammonia-dependent活动天冬酰胺合成酶(as - B气体分离器)和一系列的突变体。与amidotransferases purF家族的其他成员,重组酶具有NH2-terminal半胱氨酸残基。更换Cys-1丙氨酸和丝氨酸导致的损失谷氨酰胺酶和glutamine-dependent活动,没有任何重大影响ammonia-dependent天冬酰胺的合成。如前所观察到的人类(盛,S。Moraga-Amador D。范Heeke, G。艾莉森,r D。理查兹,n . g . J。舒斯特尔,s m(1993)生物。化学。268年,16771 - 16780),谷氨酰胺是一种抑制剂的ammonia-dependent反应催化Cys-1 - - > Ala (C1A)和Cys-1 - - > Ser (c1)的突变体,as - b气体分离器。对于C1A,抑制模式表明,一个失败的复杂的形成。 This is consistent with a recent proposal implicating the formation of an imide intermediate in the nitrogen transfer reaction (Richards, N. G. J., and Schuster, S. M. (1992) FEBS Lett. 313, 98-102). In contrast, glutamine appears to be only a competitive inhibitor of the ammonia-dependent activity of C1S. Cys-1 does not appear to be required for glutamine binding. Replacement of Asp-33 by either asparagine or glutamic acid has little effect on the kinetic properties of the mutant enzymes when compared to wild-type AS-B. Cys-1 and Asp-33 are cognate to residues Cys-1 and Asp-29 in glutamine phosphoribosylpyrophosphate amidotransferase which have been proposed to be members of a catalytic triad responsible for mediating nitrogen transfer in this enzyme (Mei, B., and Zalkin, H. (1989) J. Biol. Chem. 264, 16613-16619). In the case of AS-B, although Cys-1 is essential for glutamine-dependent activity, Asp-33 does not appear to participate in mediating nitrogen transfer. In an effort to locate other residues which might form part of a "catalytic triad" in the glutamine amidotransferase domain of AS-B, we have expressed and characterized mutant proteins in which His-29 and His-80, which are conserved within the glutamine amidotransferase domain of purF amidotransferases, are replaced by alanine (H29A and H80A).(ABSTRACT TRUNCATED AT 400 WORDS)

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药物靶点

药物	目标	类	生物	药理作用	行动
谷氨酸	GMP合成酶(glutamine-hydrolyzing)	蛋白质	人类	未知的	不可用	细节