Turnbull J L, Waldrop G L, Schachman H K
Department of Molecular and Cell Biology, University of California, Berkeley 94720.
Biochemistry. 1992 Jul 21;31(28):6562-9. doi: 10.1021/bi00143a028.
The chemical and kinetic mechanisms of the reaction catalyzed by the catalytic trimer of aspartate transcarbamoylase have been examined. The variation of the kinetic parameters with pH indicated that at least four ionizing amino acid residues are involved in substrate binding and catalysis. The pH dependence of K(ia) for carbamoyl phosphate and the K(i) for N-(phosphonoacetyl)-L- aspartate revealed that a protonated residue with a pK value of 9.0 is required for the binding of carbamoyl phosphate. However, the variation with pH of K(i) for succinate, a competitive inhibitor of aspartate, and for cysteine sulfinate, a slow substrate, showed that a single residue with a pK value of 7.3 must be protonated for binding these analogues and, by inference, aspartate. The profile of log V against pH displayed a decrease in reaction rate at low and high pH, suggesting that two groups associated with the Michaelis complex, a deprotonated residue with a pK value of 7.2 and a protonated group with a pK value of 9.5, are involved in catalysis. By contrast, the catalytically productive form of the enzyme-carbamoyl phosphate complex, as illustrated in the bell-shaped pH dependence of log (V/K)(asp), is one in which a residue with a pK value of 7.0 must be protonated while a group with a pK value of 9.1 is deprotonated. This interpretation is supported by the results from the temperature dependence of the V and V/K profiles and from the pH dependence of pK(i) for the aspartate analogues.(ABSTRACT TRUNCATED AT 250 WORDS)
已对天冬氨酸转氨甲酰酶催化三聚体所催化反应的化学和动力学机制进行了研究。动力学参数随pH的变化表明，至少有四个可电离的氨基酸残基参与底物结合和催化过程。氨甲酰磷酸的K(ia)和N-(膦酰基乙酰基)-L-天冬氨酸的K(i)对pH的依赖性表明，氨甲酰磷酸结合需要一个pK值为9.0的质子化残基。然而，天冬氨酸的竞争性抑制剂琥珀酸以及缓慢底物半胱氨酸亚磺酸盐的K(i)随pH的变化表明，一个pK值为7.3的单一残基必须质子化才能结合这些类似物，由此推断也能结合天冬氨酸。log V对pH的曲线显示在低pH和高pH下反应速率降低，这表明与米氏复合物相关的两个基团，一个pK值为7.2的去质子化残基和一个pK值为9.5的质子化基团，参与了催化过程。相比之下，酶 - 氨甲酰磷酸复合物的催化活性形式，如log (V/K)(asp)呈钟形的pH依赖性所示，是一个pK值为7.0的残基必须质子化而pK值为9.1的基团去质子化的形式。V和V/K曲线的温度依赖性结果以及天冬氨酸类似物pK(i)的pH依赖性结果支持了这一解释。（摘要截短于250字）
