Regulation of phosphatidate phosphatase activity from the yeast Saccharomyces cerevisiae by phospholipids

Regulation of Saccharomyces cerevisiae membrane-associated phosphatidate phosphatase (3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4) activity by phospholipids was examined using purified enzyme and Triton X-100/phospholipid-mixed micelles, Anionic phospholipids activated phosphatidate phosphatase activity whereas zwitterionic phospholipids had a slight inhibitory effect on activity. Cardiolipin (A(0.5) = 1.9 mol %), CDP-diacylglycerol (A(0.5) = 2.6 mol %), and phosphatidylinositol (A(0.5) = 5.5 mol %) were the most potent anionic phospholipid activators, Enzyme activation by cardiolipin (n = 2.8), CDP-diacylglycerol (n = 2.1), and phosphatidylinositol (n = 3.3) followed positive cooperative kinetics. A kinetic analysis was performed to determine the mechanism of phosphatidate phosphatase activation by anionic phospholipids. The dependence of phosphatidate phosphatase on phosphatidate was cooperative (n similar to 2.2) in the absence and presence of phospholipid activators. Cardiolipin, CDP diacylglycerol, and phosphatidylinositol were mixed competitive activators of phosphatidate phosphatase activity. The major effect of the activators was to cause a decrease in the K-m for phosphatidate. Sphinganine, a positively charged sphingoid base, inhibited phosphatidate phosphatase activity and antagonized the activation of the enzyme by cardiolipin and phosphatidylinositol. Sphinganine caused an increase in the cooperativity of cardiolipin activation, but had litt le effect on the A(0.5) value for cardiolipin. On the other hand, sphinganine had little effect on the cooperativity of phosphatidylinositol activation, but caused an increase in the A(0.5) value for phosphatidylinositol. The activation constants for cardiolipin, CDP-diacylglycerol, and phosphatidylinositol were within the range of their cellular concentrations. These results suggested that the activation of phosphatidate phosphatase activity by anionic phospholipids may be physiologically relevant.