Javid-Majd F, Stapleton M A, Harmon M F, Hanks B A, Mullins L S, Raushel F M
Department of Chemistry, Texas A&M University, College Station 77843, USA.
Biochemistry. 1996 Nov 12;35(45):14362-9. doi: 10.1021/bi961184q.
Carbamoyl phosphate synthetase (CPS) from Escherichia coli catalyzes the formation of carbamoyl phosphate from two molecules of MgATP, bicarbonate, and glutamine. It has been previously shown that the amino- and carboxy-terminal halves of the large subunit of this protein are homologous. A working model for the active site structure of the carboxy-terminal domain of the large subunit of CPS was constructed based upon amino acid sequence alignments and the previously determined three-dimensional structures of two mechanistically related proteins, biotin carboxylase and D-alanine:D-alanine ligase. The model was tested by mutation of ten amino acid residues predicted to be important for binding and/or catalysis. The mutated residues were as follows: R571, R675, R715, D753, E761, N827, Q829, E841, N843, and R845. The mutant proteins were expressed, purified to homogeneity and the catalytic properties determined for a variety of assay formats. The mutants E761A, E841Q, N843D, and R845Q were diminished in their ability to synthesize carbamoyl phosphate. The R715A, Q829A, and R675A mutants displayed elevated Michaelis constants for MgADP in the partial back reaction. The mutants E761A, N827A, E841Q, N843D, and R845Q showed significant increases in the Michaelis constants for either bicarbonate or carbamoyl phosphate. No significant alterations were noted upon mutation of either R571 or D753 to an alanine residue and thus these amino acids do not appear essential for structure or catalytic activity. These results have been utilized to further support the proposal that the C-terminal half of the large subunit of CPS is primarily responsible for the phosphorylation of the carbamate intermediate during the final formation of carbamoyl phosphate. The measured effects on the catalyic activities displayed by these mutations were found to be comparable to the previously determined effects after mutation of the homologous residues located on the N-terminal half of CPS and also for those residues mutated within D-alanine:D-alanine ligase [Shi, Y., & Walsh, C.T. (1995) Biochemistry 34, 2768-2776].
来自大肠杆菌的氨甲酰磷酸合成酶（CPS）催化由两分子MgATP、碳酸氢盐和谷氨酰胺形成氨甲酰磷酸。先前已经表明，该蛋白质大亚基的氨基末端和羧基末端部分是同源的。基于氨基酸序列比对以及先前确定的两种机制相关蛋白质（生物素羧化酶和D-丙氨酸：D-丙氨酸连接酶）的三维结构，构建了CPS大亚基羧基末端结构域活性位点结构的工作模型。通过对预测对结合和/或催化重要的十个氨基酸残基进行突变来测试该模型。突变的残基如下：R571、R675、R715、D753、E761、N827、Q829、E841、N843和R845。表达突变蛋白，纯化至同质，并针对多种测定形式测定催化特性。突变体E761A、E841Q、N843D和R845Q合成氨甲酰磷酸的能力降低。R715A、Q829A和R675A突变体在部分逆向反应中对MgADP的米氏常数升高。突变体E761A、N827A、E841Q、N843D和R845Q对碳酸氢盐或氨甲酰磷酸的米氏常数显著增加。将R571或D753突变为丙氨酸残基后未观察到明显变化，因此这些氨基酸对于结构或催化活性似乎不是必需的。这些结果已被用于进一步支持以下提议：CPS大亚基的C末端部分在氨甲酰磷酸最终形成过程中主要负责氨基甲酸酯中间体的磷酸化。发现这些突变对催化活性的测量影响与先前在CPS N末端部分的同源残基突变后以及在D-丙氨酸：D-丙氨酸连接酶中突变的那些残基后确定的影响相当[Shi，Y.，& Walsh，C.T.（1995）Biochemistry 34，2768 - 2776]。
