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

The spectrum of fatty acids produced by the fatty acid synthetase complex of Mycobacferium phZei 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 ((214 to C18) to longer chain acids (C,, to CQ~) 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 pM acetyl-CoA) to 150 (3 mM acetyl-CoA) increases the percentage of shorter chain acids from 12% to 87%. The addition of mycobacterial 3-0methylmannose containing polysaccharide (MMP) or 6-0methylglucose containing polysaccharide (MGLP) at standard assay conditions (300 pM acetyl-CoA) causes a similar shift from a low (25 %) to a high (85 %) proportion of shorter chain acids, concurrent with a 5to lo-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 pM acetyl-CoA and CoA concentrations up to 100 pM, the formation of short chain acids is favored, whereas at higher CoA concentrations there is a shift toward the longer (C20 to C,,) acids. M. Phlei palmitoyl thioesterase when added to the standard assay system (300 PM 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 (C,, and C,,) 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 GIG-CoA (or C&-CoA) will therefore affect the synthetic rate, the product distribution or both. They may do so by competition (high concentrations of

The spectrum of fatty acids produced by the fatty acid synthetase complex of Mycobacferium phZei 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 ((214 to C18) to longer chain acids (C,, to CQ~) 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 pM acetyl-CoA) to 150 (3 mM acetyl-CoA) increases the percentage of shorter chain acids from 12% to 87%.
The addition of mycobacterial 3-0methylmannose containing polysaccharide (MMP) or 6-0methylglucose containing polysaccharide (MGLP) at standard assay conditions (300 pM acetyl-CoA) causes a similar shift from a low (25 %) to a high (85 %) proportion of shorter chain acids, concurrent with a 5-to lo-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 pM acetyl-CoA and CoA concentrations up to 100 pM, the formation of short chain acids is favored, whereas at higher CoA concentrations there is a shift toward the longer (C20 to C,,) acids.
M. Phlei palmitoyl thioesterase when added to the standard assay system (300 PM acetyl-CoA) 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 (C,, and C,,) 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 GIG-CoA (or C&-CoA) will therefore affect the synthetic rate, the product distribution or both. They may do so by competition ( The fatty acid synthetase complex from Mycobacterium phlei produces CoA derivatives of saturated straight chain fatty acids 14 to 26 carbon atoms long (1). The prominent feature of the .I[. phlei system is that it affords not only fatty acids of tin unusual length, but also that their distribution pattern is bimodal. Palmitate and tetracosanoate predominate and C14, Cl*, C20, arid CzZ are found in lesser amounts.
It has been established that this bimodal pattern is due to the activity of a single homogeneous multienzymc complex and not due to the combined activities of two separate synthetasc systems (2).
Several other noteworthy properties of the Al. phlei synthetase complex have been reported.
Unusually high concentrations of acctyl-CoA and the presence of one of the mycobacterial polysaccharides MMl" or MGLl' are needed for optimal activity (3). The synthetase also catalyzes the elongation of palmitoyl-Coh or stearoyl-CoA to CZ2 and CZ4 acids (1) but this process requires, in addition to polysaccharide, the presence of free CoA or of a thioesterase which produces free CoA from the primer substrate (4). We now show that the spectrum of enzymatically produced fatty acids can be drastically altered by varying the conditions for assaying the synthetase.
The pattern can be shifted either toward very low or very high relative proportions of shorter chain (Cl4 to Cl,) and longer chain (C&o to C24) acids. Carrier fatty acid methyl esters (C14, C16, C18, C&O, C&, and C&d) were purchased from Supelco, Inc.
Methods-The :II. phlei fatty acid synthetase complex, the mycobacterial polysaccharides MMP and MGLP-I and palmitoyl thioesterase were isolated and purified as previously described (3,5). Methylated MGP was prepared as described by Vance et al. (2).
Enzyme (4 pg) was added to start the reactions which were terminated after 15 min at 37" by the addition of 0.15 ml of 50% KOH.
Carrier fatty acid methyl esters (50 pg of a mixture of Cl4 to CZ4) were then added to each tube and the mixtures heated on a boiling water bath for 20 min. After acidification with 6 N HCl, fatty acids were extracted with two 5-ml portions of petroleum ether. The solvent was transferred to screwcap culture tubes and evaporated under a stream of nitrogen.
For converting the acids to methyl esters 1 ml of 0.75 N HCl in methanol, prepared by bubbling HCl gas into methanol was added and the tubes were capped (Teflonlined caps) and heated at 75" overnight in a heating block. Solid silver carbonate was added to neutralize the acid and the methanol solution transferred to vials and evaporated under nitrogen. The residue was taken up in a small amount of hexane and assayed for total radioactivity prior to gas chromatographic analysis.
Methyl esters were analyzed by gas-liquid chromatography on a 6 ft. column of 12% stabilized diethyleneglycol succinate with an F & M model 400 instrument equipped with a hydrogen flame detector and variable stream splitter.
The column temperature was 160" for Cl4 to Cl* esters and 180" for CZO to C24 esters. Effluent was either passed directly into a proportional counter (Nuclear Chicago) with methane as the carrier, or burned to CO2 and water and passed into a proportional counter (Packard model 894) with propane as carrier.
The values for each acid are expressed as percentages of the total radioactivity in the sample.

RESULTS
Under standard conditions the ill. phlei synthetase is assayed in the presence of relatively high acetyl-CoA concentrations because the K, for acetyl-CoA is very large in this system (2) unless one of the mycobacterial polysaccharides is added. At 300 pM acetyl-CoA and 20 pM malonyl-CoA and in the absence of either MMP or MGLP, the enzyme produces the fatty acid spectrum shown in Fig. 1. Under these conditions longer chain acids (CzZ to C&4) account for about 70% of the total and the shorter acids (Cl4 to Cl,) for the remainder.
The most critical determinant of chain length as a function of substrate concentration appears to be the ratio of acetyl-CoA to malonyl-CoA rather than the absolute concentration of acetyl-CoA. As the ratio of these two substrates is increased from 0.40 (acetyl-CoA, 20 PM; malonyl-CoA, 50 PM) to 150 (acetyl-CoA, 3 mM; malonyl-CoA, 20 PM) the percentage of shorter chain acids rises from a low of 129$ to a high of 87% (Figs. 2, 3, and 4). It should be noted that at the highest acetyl-CoA to malonyl-CoA ratio chosen the concentration of acetyl-CoA is 3 mM and that under these conditions the over-all rate of synthesis ([14C]malonyl-CoA incorpora-tion) is the same as that obtained with 300 PM acetyl-CoA and optimal concentrations of polysacchraide. Polysaccharide Eflects-At an acetyl-CoA concentration of 300 pM the addition of graded amounts of mycobacterial polysaccharides progressively raises the proportion of shorter chain acids (Fig. 5). The latter comprise 85% of the total with optimal amounts of MMP and 80% with optimal MGLP-I.
Palmitate, which increases from 15y0 to 60% under these conditions, accounts for most of the polysaccharide-induced increase in this group of shorter chain acids (Fig. 6). In the absence of polysaccharide the proportion of the Cl4 to Cl8 acids is only 25% (Fig. 1). As pointed out above, optimal polysaccharide affects the chain length pattern and the over-all synthetic rate in much the same way as do very high acetyl-CoA to malonyl-CoA ratios suggesting a common or similar mode of action.
MGLP, a polysaccharide containing both glucose and 6-Omethylglucose and various acyl groups in sugar ester linkage, can be deacylated to a product designated as MGl' (2). When treated with dimethylsulfate, MGP yields a methylated product which is only one-third as effective as MGLP in stimulating over-all fatty acid synthesis (2). Methylated MGP also raises the relative proportion of the shorter chain acids but the effect is much smaller than with MGLP-I or MMP (Fig. 5). EJects of BSA-BSA stimulates the rate of the reactions catalyzed by the fatty acid synthetase complexes of yeast and of Corynebacterium diphtheriae and enhances in these systems the accumulation of palmitate at the expense of stearate (6). Although BSA is without effect on over-all X. ph2ei synthetase activity, it profoundly alters the spectrum of the fatty acids produced.
At 300 pM acetyl-CoA, the proportion of shorter chain acids rises from 257, of the total in the absence of BSA to 70% in its presence (1 .O mg per ml) (Fig. 7). However, even at the highest levels of BSA tested (4 mg per ml) the longer chain acids (C& continue to be synthesized. Effects of CoA and Palmitoyl-CoA Thioesterase on Synthetase Rate and Fatty Acid Spectrum-Free CoA was previously found to be essential for supporting palmitoyl-CoA elongation as catalyzed by the Jf. phlei synthetase (4). When the enzyme is assayed for de novo synthesis with 50 pM acetyl-CoA as the primer and in the absence of polysaccharide, 500 pM CoA will stimulate the synthetic rate by as much as 4-fold (Fig. 8)

DISCUSSION
The regulation of the JI. ph2ei synthetase activity poses a number of problems that are not encountered with functionally analogous multienzyme complexes from other sources. The apparently unique properties of the Jr. phlei system include (a) the unusually high K, for acetyl-CoA (1); (b) the requirement for DI'NH as well as Tl'NH due to the different pyridine nucleotide specificities of the P-keto-acyl-reductase and the (Y, /3-enoyl-reductase steps (7)  Among these features of the YV. phlei system two seem to us of central interest.
They are the mechanism underlying the effects of polysaccharides on enzyme activity and the bimodal fatty acid pattern. Conceivably these two phenomena are related.
In order to shed more light on these regulatory aspects, we have now examined the factors that determine the JI. phlei fatty acid chain length pattern.
The spectrum is highly variable but persistently bimodal with one maximum at Cl6 or Cl8 and another at C&d. Obviously, the relative abundance of acids of different chain length is determined by the activities and specificities of the enzymes which terminate growth of the fatty acid chain. The problem, chain termination in fatty acid synthesis catalyzed by multienzyme complexes, has been analyzed in detail for the yeast enzyme by Sumper et al. (8). One of the variables tested was the rat#io of acetyl-CoA to malonyl-CoA.
When this ratio was abnormally low, stearate was the principal product, whereas at high substrate ratios, palmitate predominated and the shorter chain acids also tended to increase.
All observed changes in the proportion of longer and shorter chain end products were con- sistent with a proposed model according to which chain length is determined by the relative velocities of the condensing enzyme which lengthens the chain and the terminal transacylase which discharges the fatty acid from the complex.
Since the nr. phlei product spect,rum is bimodal and since each mode consists of one major and of several minor acids, we shall consider any shifts in the distribution pattern in terms of the two groups, shorter (S, Cl4 to CJ and longer (L, CZo to C2,) acids. Changes in the acetyl-CoA to malonyl-CoA ratio affect the Jr.
phl ei synthetasc in the expected fashion. The higher the ratio the short)er the length of the fatty acids produced.
At one extreme when the substrate ratio [acctyl-CoA to malonyl-CoA] is 0.4, t,he L component amounts to 85 to 90% of the total, and at the other, with a 150 molar excess of acctyl-CoA, the S:L rat,io is the reverse, the S group accounting for 80% of the total. Thus, the direct,ion of the change in response to varying substrate rat,ios is the same for the IIf. phlei sgnthetase as for the yeast enzyme (8) escept that in the mycobacterial system one observes relative increases or decreases of groups of fatty acids differing in length by 6 to 8 carbon atoms, whereas in yeast the relative proportions of neighboring fatty acids (e.g. Cl0 and C,,) are affected. There is good evidence that the malonyl transacylase and palmitoyl transacylase component)s of the yeast synthetase are ident,ical (10) and this seems to be t,rue also for the JI. phl ei system.3 Therefore, malonyl-CoA at very high concentration may competitively inhibit palmitoyl transacylation and favor chain extension beyond Ci6. This will result in an increase of the longer chain acids at the expense of palmitatc.
DSA causes the synthetases of yeast and Corynebacteriu?n di phtheriae to produce more palmitate and less stearate (6). In the presence of USA the fatty acid spectrum of the X. phl ei enzyme also shifts in favor of shorter acids. The effect is much more pronounced in rlf. phl ei than in the two other microbial systems. Moreover, the principal change is in the concentration of Cl6 relative to CZ4 rather than of Cl6 relative to C18. Early chain termination in the presence of llSA can be attributed to sequestration of fatty acylKoA by protein on the assumption that the strength of the interaction increases with the length of fatty acyl-CoA and that it reaches a maximum at C&. Removal of product in the complexed form will shift the equilibrium of the reversible transacylase reaction toward palmitoyl-CoA production and thereby retard the further extension of Cl6 chains.
The two mycobacterial polysaccharides MGLP and MMP have recently been shown to duplicate the effects of I%A on fatty acid synthesis catalyzed by the enzyme from yeast and Corynebacterium (6). They also cause an increase in the production of myristate and palmitate relative to stearate in these systems. On the basis of these observations it was proposed (6) and then demonstrated that MMI' and MGLP, like ISSA, form adducts with acyl-CoA derivatives of the appropriate chain length (11).
The product pattern of the !I[. phlei fatty acid synthctase is very markedly altered by MMP or MGLP.
In the absence of MMl' or MGLP, C24 predominates (8OLjb of total) while in their presence, the shorter acids (C,, to C,,) account for 80% of the total.
The very similar alterations in the 111. phlei acid spectrum caused by MM1 or MGLP on the one hand and by 13SA on the other can both be explained by palmitoyl-CoA binding which in turn will shift the transacylase equilibrium.
However, as previously noted, IBA does not share the property of MXII' or AfGLl' to stimulate the over-all rate of ~11. phl ei synthetase reactions (1). This dissimilar response is also evident under various other circumstances. For example, &f. phlei synthetase assayed with 300 pM acetyl-CoA and in the absence of factors is completely inhibited by 15 WM palmitoyl-Cob.
Uoth BSA and MMP or MGLP relieve this inhibition but whereas BSA restores activity only to .the normal level, the polysaccharides raise enzyme activity several fold above that of the controls (Fig. 9). Secondly, elongation of palmitoyl-CoA or stearoyl-CoA by the M. phlei synthetase requires MMP or MGLP (4). Under these conditions BSA is inactive.
It is clear, therefore, that the mycobacterial polysaccharides, while sharing with BSA certain regulatory properties the basis of which is long chain acyl-CoA binding, MMP and MGLP exert additional and more specific effects on Al. phlei fatty acid synthesis not explainable on that basis.4 Attempts to pinpoint these additional effects of MMP or MGLP have included studies of the various partial reactions catalyzed by the Jl. phlei synthetase (acetyl-and malonyltransacylation, condensation, etc.) but none of these individual events are significantly stimulated or inhibited by MMP or MGLP.5 A working hypothesis for the mode of action of the mycobacterial polysaccharides is outlined in Fig. 10. First, in order to explain the bimodal fatty acid pattern, we postulate the existence in the dl. phlei complex of two long chain transacylases catalyzing the reversible reaction acyl-E + CoA s acyl-CoA + E (1) The first of the two long chain transacylases would operate optimally at the Cl6 level and the second at C&. It is proposed that the over-all rate and the product pattern (Cl, versus C,,)  is controlled by the relative activities of the two long chain transacylases and also by the activity of the condensing enzyme.
The chain shortening effects of both BSA and MMP or MGLP are readily explained by adduct formation between the complexing molecule and palmitoyl-Cod once this product has dissociated from the enzyme.
The relief of inhibition by external palmitoyl-CoA is similarly explained. Complex formation will shift the equilibrium of the reversible Cl6 transacylase (Reaction 1) in favor of free palmitoyl-CoA and, therefore, compete with the enzyme activities that lengthen the Cl6 chain. In order to rationalize the specific effects of the polysaccharides on over-all reaction rates, we propose that chain termination at the Cl6 level is the rate limiting step. A fatty acid chain that has grown to this length will be removed from the multienzyme complex by the following events (13-16) : Cls-S-ACP-E ti Cle-Ser-E CoA y C,&oA + E We further assume that C&S-ACP-E or C&er-E are accessible to and available for interaction with MMP or MGLP. If the formation of such complexes facilitates removal of palmitoyl residues from the enzyme, perhaps by weakening hydrophobic interactions, the effect would be to accelerate the over-all rate of synthesis.6 These ad hoc assumptions are made to offer an explanation not only for the enhancement of over-all synthesis by MMP or MGLP but also for the failure of BSA to act similarly. BSA, a very much larger molecule than MMP or MGLP, may not have access to enzyme-bound palmitoyl chains. If this is so, it will form complexes only with palmitoyl-Coil that is free after release of this product from the enzyme.
Since this event is subsequent to the rate-limiting (transacylase) step, BSA will not change the over-all rate of synthesis.
The above scheme may also help to clarify the previous finding that the excessively high K, for acetyl-CoA in the M. phlei synthetase system can be drastically lowered by polysaccharide (from 900 to 80 pM) (2). We suggest that acetyl-CoA, at very high concentrations (or high acetyl-CoA to malonyl-CoA ratios) may compete with Cl6 chains for a common site. This competition would have the effect of accelerating Cl6 transacylation, increasing the proportion of shorter acids and stimulating the over-all rate of synthesis. Raising the acetyl-CoA concentration will then become equivalent to fortifying the system with polysaccharide at low acetyl-CoA concentrations, both with respect to over-all rate and alteration of the fatty acid pattern. At 3 mM acetyl-CoA the synthetase does in fact exhibit the same 6  fatty acid pattern and activity as with lower levels of acetyl-CoA (300 MM) and optimal amounts of polysaccharide.

Effects of Coenzyme A-and Palmitoyl-CoA
Thioesterase-The response of the M. phlei synthetase to free CoA is complex and depends on the concentrations of acetyl-CoA as well as of CoA. At 300 PM acetyl-CoA the coenzyme neither stimulates the overall synthetic rate nor alters the fatty acid spectrum.
When the acetyl-CoA concentration is lowered to 50 PM, optimal levels of CoA (500 PM) produce a 4-fold increase in the rate of de novo synthesis and at 1 mM CoA the relative proportion of the long chain acids rises to about 90%, the highest observed under any experimental conditions. This result is consistent with the earlier finding that CoA is limiting in chain elongation of palmitoyl-CoA to C&d acids, presumably because the transacylase operating at the C&4 level has a high K, for this coenzyme (4). CoA is the only reagent found so far which under certain specified conditions stimulates the over-all rate of de novo synthesis without affecting the chain length pattern.
A palmitoyl-CoA thioesterase, earlier referred to as elongating factor (EF, (4)), was shown to be necessary for supporting the chain extension of palmitoyl-CoA or stearoyl-Cob to G4 acids (4). Since free CoA had the same effect on elongation, it was concluded that the thioesterase functioned by hydrolyzing some of the substrate, thereby furnishing free CoA. The changes seen on addition of thioesterase to the de novo synthetase assay system can be explained on the same basis. Removal of released palmitoyl-or stearoyl-CoA by hydrolysis will shift the equilibrium of the palmitoyl transacylase in the direction of product formation favoring early chain termination.
The specificity of the thioesterase (4) is consistent with these effects. Activity is highest with palmitoyl-Cob and stearoyl-Cob. In summary, we describe here experimental conditions resulting in drastically altered product patterns of the Al. phlei fatty acid synthetase complex.
The synthetase activity can be modulated to furnish almost exclusively either the shorter (Cl, to C,,) or the longer (C& to C&4) acids and this can be achieved either with or without change in the over-all rate. The bimodal character of the fatty acid spectrum persists under all experimental conditions.
A specific acyl-CoA transacylase active with acyl chains longer than Cl8 may be responsible for some of the singular properties of the X. phlei synthetase.
To what extent the various factors are important for regulating the activity and specificity of the synthetase under physiological conditions is difficult to assess. It seems reasonable to assume, however, that the intracellular polysaccharides which are produced in relatively large amounts by the 111. phlei cell (3, 17, 18) play a major role in controlling fatty acid synthesis in this organism.
In enzyme systems that produce or utilize palmitoyl-CoA or stearoyl-CoA, the tendency of these surface-active compounds to form micelles must be taken into account.
Moreover, the values for the critical micelle concentration of a given acyl-CoA derivative vary greatly as a function of ionic strength, pH, and perhaps other factors (19). However, calculation shows that under the assay conditions for de novo fatty acid synthesis which have been examined in the present investigation, the concentration of acyl-CoA product remains far below the critical micelle concentration. It therefore appears that the variations in fatty acid spectrum described here are unrelated to this phenomenon.