Regulation of phosphatidate phosphatase activity from the yeast Saccharomyces cerevisiae by nucleotides.
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.
Department of Food Science, Cook College, New Jersey Agricultural Experiment Station, Rutgers University, New Brunswick 08903.
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Carman
D001665:Binding Sites; D005947:Glucose; D007700:Kinetics; D050356:Lipid Metabolism; D008274:Magnesium; D009711:Nucleotides / Q000494:pharmacology*; D010711:Phosphatidate Phosphatase / Q000187:drug effects / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D009711:Nucleotides; D010711:Phosphatidate Phosphatase; D008274:Magnesium; D005947:Glucose