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首页> 外文期刊>Biochemistry >Deconstruction of the catalytic array within the amidotransferase subunit of carbamoyl phosphate synthetase.
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Deconstruction of the catalytic array within the amidotransferase subunit of carbamoyl phosphate synthetase.

机译：解构氨基甲酸酯磷酸合成酶酰胺转移酶亚基内的催化阵列。

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Carbamoyl phosphate synthetase from Escherichia coli catalyzes the formation of carbamoyl phosphate from bicarbonate, glutamine, and two molecules of ATP. The enzyme consists of a large synthetase subunit, and a small amidotransferase subunit, which belongs to the Triad family of glutamine amidotransferases. Previous studies have established that the reaction mechanism of the small subunit proceeds through the formation of a gamma-glutamyl thioester with Cys-269. The roles in the hydrolysis of glutamine played by the conserved residues, Glu-355, Ser-47, Lys-202, and Gln-273, were determined by mutagenesis. In the X-ray crystal structure of the H353N mutant, Ser-47 and Gln-273 interact with the gamma-glutamyl thioester intermediate [Thoden, J. B., Miran, S. G., Phillips, J. C., Howard, A. J., Raushel, F. M., and Holden, H. M. (1998) Biochemistry 37, 8825-8831]. The mutants E355D and E355A have elevated values of K(m) for glutamine, but the overall carbamoyl phosphate synthesis reaction is unperturbed. E355Q does not significantly affect the bicarbonate-dependent ATPase or glutaminase partial reactions. However, this mutation almost completely uncouples the two partial reactions such that no carbamoyl phosphate is produced. The partial recovery of carbamoyl phosphate synthesis activity in the double mutant E355Q/K202M argues that the loss of activity in E355Q is at least partly due to additional interactions between Gln-355 and Lys-202 in E355Q. The mutants S47A and Q273A have elevated K(m) values for glutamine while the V(max) values are comparable to that of the wild-type enzyme. It is concluded that contrary to the original proposal for the catalytic triad, Glu-355 is not an essential residue for catalysis. The results are consistent with Ser-47 and Gln-273 playing significant roles in the binding of glutamine.
机译：来自大肠杆菌的氨基甲酸酯磷酸合成酶催化由碳酸氢盐，谷氨酰胺和两个ATP分子形成的氨基甲酸酯磷酸。该酶由一个大的合成酶亚基和一个小的酰胺基转移酶亚基组成，属于谷氨酰胺酰胺基转移酶的三联体家族。先前的研究已经确定了小亚基的反应机理是通过与Cys-269形成γ-谷氨酰硫酯而进行的。通过诱变确定了保守残基Glu-355，Ser-47，Lys-202和Gln-273在谷氨酰胺水解中的作用。在H353N突变体的X射线晶体结构中，Ser-47和Gln-273与γ-谷氨酰硫酯中间体相互作用[Thoden，JB，Miran，SG，Phillips，JC，Howard，AJ，Raushel，FM和Holden ，HM（1998）Biochemistry 37，8825-8831]。突变体E355D和E355A的谷氨酰胺K（m）值升高，但总的氨基甲酰磷酸合成反应不受干扰。 E355Q不会显着影响碳酸氢盐依赖性ATPase或谷氨酰胺酶的部分反应。然而，这种突变几乎完全解开了两个部分反应，从而不产生氨基甲酸酯磷酸酯。双重突变体E355Q / K202M中氨基甲酸酯磷酸酯合成活性的部分恢复认为，E355Q中活性的丧失至少部分是由于E355Q中Gln-355和Lys-202之间的额外相互作用。突变体S47A和Q273A的谷氨酰胺K（m）值升高，而V（max）值与野生型酶相当。结论是，与最初提出的催化三联体相反，Glu-355不是催化的必需残基。结果与Ser-47和Gln-273在谷氨酰胺结合中起重要作用是一致的。

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