Enzymology of cottonseed microsomal N-acylphosphatidylethanolamine synthase: Kinetic properties and mechanism-based inactivation

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• 作者：McAndrew ; Rosemary S. ; Chapman ; Kent D.
• 关键词：Acyltransferase ; Fatty acid ; Phosphatidylethanolamine ; Membrane protein ; Phospholipid ; DEPC ; diethylpyrocarbonate ; DFP ; diisopropylfluorophosphate ; DOPE ; dioleoyl(C18 ; 1 ; C18 ; 1[cis]-9)-PE ; DTNB ; dithionitrobenzoate ; DDM ; β ; -D-dodecylmaltoside ; FFA ; free
• 刊名：Biochimica et Biophysica Acta (BBA)/Lipids and Lipid Metabolism
• 出版年：1998
• 出版时间：February 5, 1998
• 年：1998
• 卷：1390
• 期：1
• 页码：21-36
• 全文大小：1.29 M

文摘

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 μmol 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 (-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 -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 -dipalmitoylphosphatidylcholine nor -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 -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.