Enzymological properties of the LPP1-encoded lipid phosphatase from Saccharomyces cerevisiae

The product of the LPP1 gene in Saccharomyces cerevisiae is a membrane-asso ciated enzyme that catalyzes the Mg2+-independent dephosphorylation of phos phatidate (PA), diacylglycerol pyrophosphate (DGPP), and lysophosphatidate (LPA). The LPP1-encoded lipid phosphatase was overexpressed 681-fold in Sf- 9 insect cells and used to examine the enzymological properties of the enzy me using PA, DGPP, and LPA as substrates. The optimum pH values for PA phos phatase, DGPP phosphatase, and LPA phosphatase activities were 7.5, 7.0, an d 7.0, respectively. Divalent cations (Mn2+, Co2+, and Ca2+), NaF, heavy me tals, propranolol, phenylglyoxal, and N-ethylmaleimide inhibited the PA pho sphatase, DGPP phosphatase, and LPA phosphatase activities of the enzyme. T he inhibitory effects of N-ethylmaleimide and phenylglyoxal on the LPP1-enc oded enzyme were novel properties when compared with other Mg2+-independent lipid phosphate phosphatases from S. cerevisiae and mammalian cells. The L PP1-encoded enzyme exhibited saturation kinetics with respect to the surfac e concentrations of PA (K-m = 0.05 mol%), DGPP (K-m = 0.07 mol%), and LPA ( K-m = 0.08 mol%). Based on specificity constants (V-max/K-m), the order of substrate preference was PA (4.2 units/mg/mol%)> DGPP (3.5 units/mg/mol%)> LPA (1.3 units/mg/mol%). DGPP (K-i = 0.12 mol%) was a competitive inhibitor with respect to PA, and PA (K-i = 0.12 mol%) was a competitive inhibitor w ith respect to DGPP. This suggested that the binding sites for these substr ates were the same. The enzymological properties of the LPP1-encoded enzyme differed significantly from those of the S. cerevisiae DPP1-encoded lipid phosphatase, a related enzyme that also utilizes PA, DGPP, and LPA as subst rates. (C) 2000 Elsevier Science B.V. All rights reserved.