Purification and characterization of fatty acyl-acyl carrier protein synthetase from Vibrio harveyi.
A Vibrio harveyi enzyme which catalyzes the ATP-dependent ligation of fatty acids to acyl carrier protein (ACP) has been purified 6,000-fold to apparent homogeneity by anion-exchange, gel filtration, and ACP-Sepharose affinity chromatography. Purified acyl-ACP synthetase migrated as a single 62-kDa band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and as an 80-kDa protein by gel filtration under reducing conditions. Activity of the purified enzyme was lost within hours in the absence of glycerol and low concentrations of Triton X-100. Acyl-ACP synthetase exhibited Kms for myristic acid, ACP, and ATP of 7 microM, 18 microM, and 0.3 mM, respectively. The enzyme was specific for adenine-containing nucleotides, and AMP was the product of the reaction. No covalent acyl-enzyme intermediate was observed. Enzyme activity was stimulated up to 50% by iodoacetamide but inhibited > 80% by N-ethylmaleimide: inhibition by the latter was prevented by ATP and ACP but not myristic acid. Dithiothreitol and sulfhydryl-directed reagents also influenced enzyme size, activity, and elution pattern on anion-exchange resins. The function of acyl-ACP synthetase has not been established, but it may be related to the capacity of V. harveyi to elongate exogenous fatty acids by an ACP-dependent mechanism.
Department of Pediatrics, Dalhousie University, Halifax, Nova Scotia, Canada.
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D000213:Acyl Carrier Protein / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D019730:Carbon-Sulfur Ligases; D004229:Dithiothreitol / Q000494:pharmacology; D004795:Enzyme Stability; D005033:Ethylmaleimide / Q000494:pharmacology; D064429:Fatty Acid Synthases / Q000187:drug effects / Q000302:isolation & purification* / Q000378:metabolism*; D005227:Fatty Acids / Q000378:metabolism*; D005990:Glycerol / Q000494:pharmacology; D007460:Iodoacetamide / Q000494:pharmacology; D008025:Ligases / Q000302:isolation & purification* / Q000378:metabolism*; D019814:Myristic Acid; D009227:Myristic Acids / Q000378:metabolism; D017830:Octoxynol; D010770:Phosphotransferases / Q000187:drug effects / Q000302:isolation & purification* / Q000378:metabolism*; D011092:Polyethylene Glycols / Q000494:pharmacology; D013379:Substrate Specificity; D017855:Transferases (Other Substituted Phosphate Groups); D014733:Vibrio / Q000201:enzymology*
D000213:Acyl Carrier Protein; D005227:Fatty Acids; D009227:Myristic Acids; D019814:Myristic Acid; D011092:Polyethylene Glycols; D000255:Adenosine Triphosphate; D017830:Octoxynol; D064429:Fatty Acid Synthases; D010770:Phosphotransferases; D017855:Transferases (Other Substituted Phosphate Groups); C021364:holo-(acyl-carrier-protein) synthase; D008025:Ligases; D019730:Carbon-Sulfur Ligases; C018749:long-chain-fatty-acid-(acyl-carrier-protein) ligase; D005033:Ethylmaleimide; D005990:Glycerol; D004229:Dithiothreitol; D007460:Iodoacetamide