In Vitro Alterations of the Product Distribution of the Fatty Acid Synthetase from Mycobacterium phlei

Abstract

Abstract The spectrum of fatty acids produced by the fatty acid synthetase complex of Mycobacterium phlei under several conditions has been examined. The observed pattern is always bimodal, consisting of palmitate and tetracosanoate as the two principal products and of lesser amounts of myristate, stearate, arachidate, and behenate. However, the relative proportions of the shorter chain acids (C14 to C18) to longer chain acids (C20 to C24) can be varied over a wide range. Such alterations can occur either with or without changes in the over-all rate of synthesis. Raising the acetyl-CoA to malonyl-CoA ratio from 0.4 (8 µm acetyl-CoA) to 150 (3 mm acetyl-CoA) increases the percentage of shorter chain acids from 12% to 87%. The addition of mycobacterial 3-O-methylmannose containing polysaccharide (MMP) or 6-Omethylglucose containing polysaccharide (MGLP) at standard assay conditions (300 µm acetyl-CoA) causes a similar shift from a low (25%) to a high (85%) proportion of shorter chain acids, concurrent with a 5- to 10-fold increase in the rate of over-all synthesis. Bovine serum albumin (BSA) has the same effect on the fatty acid pattern as MMP or MGLP (70% shorter chain acids at 1 mg of BSA per ml) but does not stimulate over-all synthesis. The effect of free CoA depends on the concentration of both the coenzyme and of acetyl-CoA. At 50 µm acetyl-CoA and CoA concentrations up to 100 µm, the formation of short chain acids is favored, whereas at higher CoA concentrations there is a shift toward the longer (C20 to C24) acids. M. phlei palmitoyl thioesterase when added to the standard assay system (300 µm acetylCoA) roughly doubles the proportion of short chain acids but does not affect the over-all rate of synthesis. To account for the widely varying fatty acid patterns in response to experimental conditions, it is proposed that fatty acyl chains (C16 and C18) either enzyme bound or accumulating as free CoA derivatives regulate the over-all rate of synthesis, perhaps by feed-back inhibition. Reagents that lower the levels of free or enzyme bound C16-CoA (or C18-CoA) will therefore affect the synthetic rate, the product distribution or both. They may do so by competition (high concentrations of acetyl-CoA), by complexing acyl-CoA (MMP, MGLP and BSA), or by thioester hydrolysis (palmitoyl thioesterase). Explanations are offered for the ability of the polysaccharides and the failure of BSA to stimulate over-all synthesis.