In previous reports it has been shown that all of the reactions of fatty acid synthesis in Escherichia coli occur with the substrates bound in thioester linkage to the acyl carrier protein (ACP). -10 The nature of the substrate binding site of ACP is of great interest as several types of reactions occur with the substrates bound to this protein. Thus, thioesters of ACP are substrates in the condensation-decarboxylation reaction,6' 7, 10 the 2 reductions,7 8 and the enoyl hydrase reaction of fatty acid synthesis.9 In each of these reactions acyl-S-ACP derivatives are either much more reactive than the corresponding acyl-S-CoA compounds, or the latter do not react at all. Initially it was reported6 that the sulfhydryl group at the binding site of ACP is a cysteine residue. Wakil1' reported that the sulfhydryl residue is accounted for by thioethanolamine and that ACP contains one mole of f3-alanine per mole of protein. These findings have been confirmed in this laboratory. Further investigation has shown that the sulfhydryl residue is part of a covalently bound prosthetic group. This report presents experiments which establish the binding site of ACP as 4'-phosphopantetheine which is probably bound through a phosphodiester linkage to a serine residue of ACP.Materials and Methods.-2-CI4-Malonyl CoA was synthesized as described previously.'2 ACP was purified as described previously from either E. coli B or from E. coli K12.A Dowex resins were purchased from Calbiochem (Biorad). P32-orthophosphate was obtained from Oak Ridge National Laboratory. 2-C14-Malonic acid was purchased from the New England Nuclear Co. Pronase was purchased from Calbiochem. Pepsin 2 X crystallized and E. coli alkaline phosphatase
Alteration of the fatty acid composition of monolayer cultures of LM cells grown in chemically defined medium was achieved by supplementation with fatty acids complexed to bovine serum albumin. Phospholipids containing up to 40% linoleate were found in cells grown in medium containing 20 mu g of linoleate/ml. Incorporation of linoleate into phospholipids reached a plateau after 12-24 hr, and cells remained viable for at least 3-4 days. Although linoleic, linolenic, and arachidonic acids were incorporated into LM cells equally well, only the latter was elongated by these cells under these experimental conditions. Nonadecanoic acid was incorporated to a lesser extent than the polyunsaturated fatty acids. Phosphatidylcholine and phosphatidylethanolamine of LM cells had different fatty acid compositions; phosphatidylethanolamine contained more longer chain and unsaturated fatty acids. Cells were also grown in the absence of choline and presence of choline analogs such as N,N-dimethylethanolamine, N-methylethanolamine, 3-amino-1-propanol, and 1-2-amino-1-butanol. The analog phospholipids in these cells had fatty acid compositions which were intermediate between those of phosphatidylethanolamine and phosphatidylcholine of control cells grown in the presence of choline. Linoleate was found in both phosphatidylcholine and phosphatidylethanolamine of cells supplemented with linoleate. The sphingolipid fraction of these cells, however, did not contain significant amounts of linoleate. When linoleate was present in the phospholipids, compensatory decreases in the oleate and palmitoleate content of phospholipids were observed. Lowering of the growth temperature to 28 degrees produced an increase in unsaturate fatty acid content of the phospholipids. When linoleate was supplied to cells grown at 28 degrees, there was no further increase in the unsaturated fatty acid composition of the phospholipids. Using both fatty acid supplementation and lowered growth temperature, LM cell membranes can be produced which have phospholipids with vastly different fatty acid compositions.
The de novo synthesis of long-chain saturated fatty acids proceeds by the following over-all reaction:Acetyl-CoA + 7 malonyl CoA + 14 TPNII + 14 H+ --palmitate + 14 TPN+ + 8 CoA + 7 C02 + 6 H20. (1) The fact that the Coenzyme A intermediates of the 3-oxidation pathway are not intermediates ill the synthesis of fatty acids from malonyl CoA in several systems,1in addition to the finding by Lynein2 4 that the product of the condensation of acetyl CoA and malonyl CoA is protein-bound acetoacetate in the yeast synthetase suggested that all of the intermediates in fatty acid synthesis might be proteinbound. As shown by Goldman et al.,5-7 the product of the condensation of acetyl CoA and malonyl CoA in the E. coli system is acetoacetate which is bound to a heatstable protein in thiolester linkage. This protein has been called Enzyme II in earlier publications. In the presence of TPNH and a crude E. coli fraction, acetoacetyl-Enzyme II is converted to butyryl-Enzyme II.7 'Furthermore, both of these compoumids are enzylnatically converted to long-chain fatty acids and therefore have been proposed as intermediates in long-chain fatty acid synthesis.6'
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