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

Abstract

SummaryPhosphatidate 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 ceramide‐1‐phosphate. All substrates were used, but the apparent Vmax 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 Vmax and interfacial Michaelis constant (KmB) 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 Vmax and KmB 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 Ki 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.