Phosphatidate phosphatase from avocado (Persea americana) - Purification, substrate specificity and possible metabolic implications for the Kennedy pathway and cell signalling in plants

Abstract

Phosphatidate phosphatase (PAP; EC 3.1.3.4) is a pivotal enzyme in plant lipid metabolism positioned at a major branchpoint between the biosyntheses of phospholipids and triacylglycerols. The enzyme has been purified 7000-fold from the microsomes of maturing Avocado fruit. The enzyme has a subunit molecular mass, as determined on SDS-PAGE, of 49 kDa, and gel filtration studies revealed that it is monomeric. The enzyme was examined for the ability to hydrolyse sn-1,2-dioleoylglycerol-3-phosphate (PA), sn-1-oleoylglycerol-3-phosphate (LPA), sn-2-oleoylglycerol-3-phosphate (sn-2-LPA), and caramide-1-phosphate. All substrates were used, but the apparent V(max) values for ceramide-1-phosphate and sn-2-LPA were considerably lower than for PA or LPA. A kinetic analysis of the purified enzyme was conducted using PA and LPA in Triton X-100 mixed micelles according to the surface dilution kinetic model. The V(max) and interfacial Michaelis constant (Km(B)) were calculated for PA as 47 μmol min-1 mg-1 and 1.49 mol%, respectively. LPA was found to be a better substrate with a V(max) and Km(B) of 100 μmol min-1 mg-1 and 1.06 mol%, respectively. A detailed kinetic analysis of the effect of LPA on PA hydrolysis revealed LPA to be a potent competitive inhibitor of the reaction, with a K(i) of 1.4 mol%. This possibly indicates that strict metabolic channelling is in operation in the Kennedy Pathway, with very low steady state concentrations of lyso-phosphatidate with respect to phosphatidate in vivo.