Kinetic analysis of Arabidopsis phospholipase Ddelta. Substrate preference and mechanism of activation by Ca2+ and phosphatidylinositol 4,5-biphosphate.
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 Ca(2+) 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 PLDdelta from Arabidopsis. PLDdelta activity was dependent on both bulk concentration and surface concentration of substrate phospholipids in the Triton X-100/phospholipid mixed micelles. V(max), K(s)(A), and K(m)(B) values for PLDdelta toward phosphatidylcholine or phosphatidylethanolamine were determined; phosphatidylethanolamine was the preferred substrate. PLDdelta activity was stimulated greatly by phosphatidylinositol 4,5-bisphosphate (PIP(2)). Maximal activation was observed at a PIP(2) molar ratio around 0.01. Kinetic analysis indicates that PIP(2) activates PLD by promoting substrate binding to the enzyme, without altering the bulk binding of the enzyme to the micelle surface. Ca(2+) is required for PLDdelta activity, and it significantly decreased the interfacial Michaelis constant K(m)(B). This indicates that Ca(2+) activates PLD by promoting the binding of phospholipid substrate to the catalytic site of the enzyme.
Qin
Chunbo
C
Department of Biochemistry, Kansas State University, Manhattan, KS 66506, USA.
Wang
Cunxi
C
Wang
Xuemin
X
D017360:Arabidopsis / Q000201:enzymology*; D002118:Calcium / Q000378:metabolism*; D020134:Catalytic Domain; D018076:DNA, Complementary / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D004926:Escherichia coli / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D007700:Kinetics; D008823:Micelles; D008956:Models, Chemical; D017830:Octoxynol / Q000494:pharmacology; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000737:chemistry* / Q000378:metabolism; D010739:Phospholipase D / Q000737:chemistry* / Q000378:metabolism; D010743:Phospholipids / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013379:Substrate Specificity
D018076:DNA, Complementary; D003902:Detergents; D008823:Micelles; D019269:Phosphatidylinositol 4,5-Diphosphate; D010743:Phospholipids; D011993:Recombinant Fusion Proteins; D017830:Octoxynol; D005982:Glutathione Transferase; C446530:phospholipase D delta; D010739:Phospholipase D; D002118:Calcium