Kinetic Analysis of Arabidopsis Phospholipase Dδ

Phospholipase D (PLD) is a major plant phospholipase family involved in many cellular processes such as signal transduction, membrane remodeling, and lipid degradation. Five classes of PLDs have been identified in Arabidopsis thaliana, and Ca2+ and polyphosphoinositides have been suggested as key regulators for these enzymes. To investigate the catalysis and regulation mechanism of individual PLDs, surface-dilution kinetics studies were carried out on the newly identified PLDδ fromArabidopsis. PLDδ activity was dependent on both bulk concentration and surface concentration of substrate phospholipids in the Triton X-100/phospholipid mixed micelles.V max, K sA, andK mB values for PLDδ toward phosphatidylcholine or phosphatidylethanolamine were determined; phosphatidylethanolamine was the preferred substrate. PLDδ activity was stimulated greatly by phosphatidylinositol 4,5-bisphosphate (PIP2). Maximal activation was observed at a PIP2 molar ratio around 0.01. Kinetic analysis indicates that PIP2 activates PLD by promoting substrate binding to the enzyme, without altering the bulk binding of the enzyme to the micelle surface. Ca2+ is required for PLDδ activity, and it significantly decreased the interfacial Michaelis constantK mB. This indicates that Ca2+activates PLD by promoting the binding of phospholipid substrate to the catalytic site of the enzyme.