Wu W I, Carman G M
Department of Food Science, Cook College, New Jersey Agricultural Experiment Station, Rutgers University, New Brunswick 08903.
J Biol Chem. 1994 Nov 25;269(47):29495-501.
Regulation of Saccharomyces cerevisiae membrane-associated phosphatidate phosphatase (3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4) activity by nucleotides was examined using pure enzyme and Triton X-100/phosphatidate-mixed micelles. Adenosine, guanosine, cytidine, and uridine nucleotides inhibited phosphatidate phosphatase activity in a dose-dependent manner. ATP and CTP were the most potent inhibitors of the enzyme. A kinetic analysis was performed to determine the mechanism of enzyme inhibition by nucleotides. The mechanism of inhibition by ATP and CTP with respect to phosphatidate (the substrate) was complex. The dependence of phosphatidate phosphatase activity on phosphatidate was cooperative, and nucleotides affected both Vmax and Km. ATP did not inhibit phosphatidate phosphatase activity by binding to the enzyme or to phosphatidate. Phosphatidate phosphatase dependence on Mg2+ ions (the cofactor) followed saturation kinetics, and the mechanism of nucleotide inhibition with respect to Mg2+ ions was competitive. Thus, the mechanism of enzyme inhibition by nucleotides was the chelation of Mg2+ ions. The inhibitor constant for ATP was lower than its cellular concentration in glucose-grown cells. However, the inhibitor constant for ATP was higher than its cellular concentration in glucose-starved cells. Changes in the cellular concentration of ATP affected the proportional synthesis of triacylglycerols and phospholipids. These results were consistent with the regulation of phosphatidate phosphatase activity by ATP through a Mg2+ ion chelation mechanism.
利用纯酶以及Triton X-100/磷脂酸混合胶束，研究了核苷酸对酿酒酵母膜相关磷脂酸磷酸酶（3-sn-磷脂酸磷酸水解酶，EC 3.1.3.4）活性的调节作用。腺苷、鸟苷、胞苷和尿苷核苷酸以剂量依赖的方式抑制磷脂酸磷酸酶活性。ATP和CTP是该酶最有效的抑制剂。进行了动力学分析以确定核苷酸对酶的抑制机制。ATP和CTP对磷脂酸（底物）的抑制机制较为复杂。磷脂酸磷酸酶活性对磷脂酸的依赖性具有协同性，核苷酸同时影响Vmax和Km。ATP并非通过与酶或磷脂酸结合来抑制磷脂酸磷酸酶活性。磷脂酸磷酸酶对Mg2+离子（辅因子）的依赖性遵循饱和动力学，核苷酸对Mg2+离子的抑制机制具有竞争性。因此，核苷酸对酶的抑制机制是Mg2+离子的螯合作用。ATP的抑制常数低于其在葡萄糖培养细胞中的细胞浓度。然而，ATP的抑制常数高于其在葡萄糖饥饿细胞中的细胞浓度。细胞内ATP浓度的变化影响三酰甘油和磷脂的比例合成。这些结果与ATP通过Mg2+离子螯合机制对磷脂酸磷酸酶活性的调节作用一致。
