title: Enzymology of cottonseed microsomal N-acylphosphatidylethanolamine synthase: kinetic properties and mechanism-based inactivation.
abstract: An ATP-, Ca2+-, and CoA-independent acyltransferase activity, designated "N-acylphosphatidylethanolamine (NAPE) synthase", was reported to catalyze the direct acylation of phosphatidylethanolamine (PE) with free fatty acids (FFAs) in cottonseed microsomes [K.D. Chapman, T.S. Moore, Jr., Plant Physiol. 102 (3) (1993) 761-769]. Here, NAPE synthase was purified 138, 176-fold from crude cottonseed homogenates to a specific activity of 5.98 mumol min-1 mg-1 protein by immobilized artificial membrane chromatography. Enzyme purity was confirmed by the presence of a 64 kDa polypeptide in fractions analyzed by tricine-SDS-PAGE. Initial velocity measurements with various free fatty acids ([14C]-linoleic, -palmitic, -oleic, -stearic and -myristic acids) and saturating concentrations of dioleoyl-PE revealed non-Michaelis-Menten, biphasic kinetics with high and low affinity sites demonstrating positive cooperativity specific for each [14C]-FFA. In contrast to FFA substrates, no kinetic differences were observed for two different molecular species of PE, (18:1,18:1)-PE and (16:0,18:2)-PE, and biphasic curves were not pronounced. Neither [14C]-dipalmitoylphosphatidylcholine nor [14C]-palmitoyl-CoA served as acyl donors for the synthesis of NAPE, indicating a preference for FFAs as the acyl donor. Also, neither ethanolamine nor sphingosine functioned as acyl acceptor molecule to form N-acylethanolamine or ceramide, respectively, indicating specificity for the phospholipid PE. NAPE synthase was inactivated in a time- and concentration-dependent manner by diisopropylfluorophosphate (DFP) through the apparent modification of one serine residue. Palmitic acid protected the enzyme from DFP-inactivation and [14C]-DFP incorporation, suggesting that a serine residue probably binds FFAs in the enzyme's active site forming an acyl-enzyme intermediate. Collectively, these results provide new information on the kinetic behavior of a purified, integral membrane enzyme which synthesizes a bilayer-stabilizing product from two lipid-soluble substrates. The biochemical properties of cottonseed NAPE synthase are consistent with a possible free fatty acid scavenging role in vivo. (c) 1998 Elsevier Science B.V.
authors.affiliation: Division of Biochemistry and Molecular Biology, Department of Biological Sciences, University of North Texas, Denton, TX 76203-5220, USA.
authors.forename: R S
authors.initials: RS
authors.lastname: McAndrew
authors.forename: K D
authors.initials: KD
authors.lastname: Chapman
mesh_terms: D000215:Acylation; D000217:Acyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D002384:Catalysis; D005231:Fatty Acids, Unsaturated / Q000378:metabolism; D003368:Gossypium / Q000201:enzymology*; D007700:Kinetics; D010714:Phosphatidylethanolamines / Q000378:metabolism; D012639:Seeds / Q000201:enzymology
chemical_list: D005231:Fatty Acids, Unsaturated; D010714:Phosphatidylethanolamines; D000217:Acyltransferases; C042139:phosphatidylethanolamine N-acyltransferase