Miran S G, Chang S H, Raushel F M
Department of Chemistry, Texas A&M University, College Station 77843.
Biochemistry. 1991 Aug 13;30(32):7901-7. doi: 10.1021/bi00246a005.
Carbamoyl phosphate synthetase from Escherichia coli catalyzes the formation of carbamoyl phosphate from ATP, bicarbonate, and glutamine. The amidotransferase activity of this enzyme is catalyzed by the smaller of the two subunits of the heterodimeric protein. The roles of four conserved histidine residues within this subunit were probed by site-directed mutagenesis to asparagine. The catalytic activities of the H272N and H341N mutants are not significantly different than that of the wild-type enzyme. The H353N mutant is unable to utilize glutamine as a nitrogen source in the synthetase reaction or the partial glutaminase reaction. However, binding to the glutamine active site is not impaired in the H353N enzyme since glutamine is found to activate the partial ATPase reaction by 40% with a Kd of 54 microM. The H312N mutant has a Michaelis constant for glutamine that is 2 orders of magnitude larger than the wild-type value, but the maximal rate of glutamine hydrolysis is unchanged. These results are consistent with His-353 functioning as a general acid/base catalyst for proton transfers while His-312 serves a critical role for the binding of glutamine to the active site.
来自大肠杆菌的氨甲酰磷酸合成酶催化由ATP、碳酸氢盐和谷氨酰胺形成氨甲酰磷酸。该酶的酰胺转移酶活性由异源二聚体蛋白的两个亚基中较小的亚基催化。通过定点诱变将该亚基内四个保守的组氨酸残基替换为天冬酰胺，以此来探究它们的作用。H272N和H341N突变体的催化活性与野生型酶的催化活性没有显著差异。H353N突变体在合成酶反应或部分谷氨酰胺酶反应中无法利用谷氨酰胺作为氮源。然而，H353N酶与谷氨酰胺活性位点的结合并未受损，因为发现谷氨酰胺可激活部分ATP酶反应达40%，解离常数为54微摩尔。H312N突变体对谷氨酰胺的米氏常数比野生型值大2个数量级，但谷氨酰胺水解的最大速率未变。这些结果表明，His-353作为质子转移的一般酸碱催化剂发挥作用，而His-312在谷氨酰胺与活性位点的结合中起关键作用。
