Stapleton M A, Javid-Majd F, Harmon M F, Hanks B A, Grahmann J L, Mullins L S, Raushel F M
Department of Chemistry, Texas A&M University, College Station 77843, USA.
Biochemistry. 1996 Nov 12;35(45):14352-61. doi: 10.1021/bi961183y.
Carbamoyl phosphate synthetase (CPS) catalyzes the formation of carbamoyl phosphate from glutamine, bicarbonate, and 2 mol of MgATP. The heterodimeric protein is composed of a small amidotransferase subunit and a larger synthetase subunit. The synthetase subunit contains a large tandem repeat for each of the nucleotides used in the overall synthesis of carbamoyl phosphate. A working model for the three-dimensional fold of the carboxy phosphate domain of CPS was constructed on the basis of amino acid sequence alignments and the X-ray crystal structure coordinates for biotin carboxylase and D-alanine:D-alanine ligase. This model was used to select ten residues within the carboxy phosphate domain of CPS for modification and subsequent characterization of the kinetic constants for the mutant proteins. Residues R82, R129, R169, D207, E215, N283, and Q285 were changed to alanine residues; residues E299 and R303 to glutamine; and residue N301 to aspartate. No significant changes in the catalytic constants were observed upon mutation of either R82 or D207, and thus these residues appear to be nonessential for binding and/or catalytic activity. The Michaelis constant for ATP was most affected by modification of residues R129, R169, Q285, and N301. The binding of bicarbonate was most affected by the mutagenesis of residues E215, E299, N301, and R303. The mutation of residues E215, N283, E299, N301, and R303 resulted in proteins which were unable to synthesize carbamoyl phosphate at a significant rate. All of the mutations, with the exception of the N301D mutant, primarily affected the enzyme by altering the step for the phosphorylation of bicarbonate. However, mutation of N301 to aspartic acid also disrupted the catalytic step involved in the phosphorylation of carbamate. These results are consistent with a role for the N-terminal half of the synthetase subunit of CPS that is primarily directed at the initial phosphorylation of bicarbonate by the first ATP utilized in the overall synthesis of carbamoyl phosphate. The active site structure appears to be very similar to the ones previously determined for D-alanine:D-alanine ligase and biotin carboxylase.
氨甲酰磷酸合成酶（CPS）催化由谷氨酰胺、碳酸氢盐和2摩尔MgATP形成氨甲酰磷酸。这种异源二聚体蛋白由一个小的氨基转移酶亚基和一个较大的合成酶亚基组成。合成酶亚基在氨甲酰磷酸的整体合成中，对于所使用的每种核苷酸都包含一个大的串联重复序列。基于氨基酸序列比对以及生物素羧化酶和D-丙氨酸：D-丙氨酸连接酶的X射线晶体结构坐标，构建了CPS羧基磷酸结构域三维折叠的工作模型。该模型用于选择CPS羧基磷酸结构域内的10个残基进行修饰，并随后表征突变蛋白的动力学常数。将残基R82、R129、R169、D207、E215、N283和Q285替换为丙氨酸残基；将残基E299和R303替换为谷氨酰胺；将残基N301替换为天冬氨酸。R82或D207突变后，催化常数未观察到显著变化，因此这些残基对于结合和/或催化活性似乎并非必需。ATP的米氏常数受残基R129、R169、Q285和N301修饰的影响最大。碳酸氢盐的结合受残基E215、E299、N301和R303诱变的影响最大。残基E215、N283、E299、N301和R303的突变导致蛋白质无法以显著速率合成氨甲酰磷酸。除N301D突变体外，所有突变主要通过改变碳酸氢盐磷酸化步骤来影响该酶。然而，N301突变为天冬氨酸也破坏了氨基甲酸酯磷酸化所涉及的催化步骤。这些结果与CPS合成酶亚基N端一半的作用一致，该作用主要针对在氨甲酰磷酸整体合成中所使用的第一个ATP对碳酸氢盐的初始磷酸化。活性位点结构似乎与先前确定的D-丙氨酸：D-丙氨酸连接酶和生物素羧化酶的活性位点结构非常相似。
