Fatty acid synthetase. A steady state kinetic analysis of the reaction catalyzed by the enzyme from pigeon liver.

The kinetic mechanism of pigeon liver fatty acid synthetase action has been studied using steady state kinetic analysis. Initial velocity studies are consistent with an earlier suggestion that the enzyme catalyzes this reaction by a seven-site ping-pong mechanism. Although the range of substrate concentrations that could be used was limited by several factors, the initial velocity patterns showing the relationship between the substrates acetyl coenzyme CoA, malonyl-CoA, and NADPH appear to be a series of parallel lines, regardless of which substrate is varied at fixed levels of a second substrate. However, two of the substrates, acetyl-CoA and malonly-CoA, apparently exhibit a competitive substrate inhibition with respect to each other, but NADPH shows no inhibition of any kind. Product inhibition patterns suggest that free CoA is competitive versus acetyl-CoA and malonyl-CoA and is uncompetitive versus NADPH, and that NADP+ is competitive versus NADPH and uncompetitive versus acetyl-CoA or malonyl-CoA. These results are consistent with a seven-site ping-pong mechanism with intermediates covalently bound to 4'-phosphopantetheine (part of acyl carrier protein). Double competitive substrate inhibition by acetyl-CoA and malonyl-CoA is consistent with the rate equation derived for the over-all mechanism. The kinetic mechanism developed from these results is capable of explaining the formation of fatty acids from malonyl-CoA and NADPH alone (Katiyar, S. S., Briedis, A. V., and Porter, J. W. (1974) Arch. Biochem. Biophys. 162, 412-420) and also the formation of triacetic acid lactone from either malonyl-CoA alone or acetyl-CoA plus malonyl-CoA.     

Pyruvate:NADP+ oxidoreductase from Euglena gracilis: the kinetic properties of the enzyme

The kinetic properties for the native forward reaction of pyruvate:NADP+ oxidoreductase from Euglena gracilis were determined. The substrate kinetics gave a pattern of a ping-pong mechanism involving a competitive substrate inhibition of CoA against pyruvate. The Km values for pyruvate, CoA, and NADP+ were estimated to be 27, 6.6, and 28 microM, respectively, and the Ki value of CoA against pyruvate was 28 microM. CO2 inhibited noncompetitively against pyruvate and NADP+, and uncompetitively against CoA. Acetyl-CoA showed a competitive inhibition with respect to pyruvate and an uncompetitive inhibition with respect to NADP+. NADPH inhibited competitively versus NADP+, noncompetitively versus CoA, and uncompetitively versus pyruvate. The kinetic behavior is consistent with a two-site ping-pong mechanism involving the substrate inhibition. From the kinetic mechanism, it is proposed that the enzyme has two catalytic sites linked by an intramolecular electron-transport chain. One of these is a thiamine pyrophosphate-containing catalytic site which reacts with pyruvate and CoA to form CO2 and acetyl-CoA, and the other site functions in the reduction of NADP+. In contrast, when methyl viologen was used as an artificial one-electron acceptor substituting for NADP+, the reaction gave a pattern characteristic of an octa uni ping-pong mechanism involving a competitive substrate inhibition of CoA against pyruvate.     

Simple assay for the condensation component enzyme (beta-ketoacyl synthetase) of fatty acid synthetase.

A simple assay is described for estimating the activity of the condensation component enzyme (beta-ketoacyl synthetase) of the yeast fatty acids synthetase complex. The radioactivity liberated as 14CO2 from [1,3-14C]malonyl-CoA was trapped in phenethylamine and measured by liquid scintillation spectroscopy. Three enzyme-catalysed steps are involved: acetyl-CoA transacylase, malonyl-CoA transacylase and beta-ketoacyl synthetase; however, beta-ketoacyl synthetase is rate-limiting. beta-Ketoacyl synthetase activity was made independent of subsequent enzyme activities of the complex by excluding NADPH from the assay, thus blocking beta-ketoacyl reductase and preventing fatty acid synthesis. By this assay beta-ketoacyl synthetase activity was about 0.28 of the activity of the complex for fatty acid synthesis, compared with approximately 0.001 for published assays. Several pyridine nucleotides and derivatives were tested after it was discovered that NADH stimulated beta-ketoacyl synthetase activity to a greater extent than could be accounted for by its reactivity in providing a pathway from acetoacetyl-enzyme to fatty acid synthesis. Presumably, the release of acetoacetate from the central sulphydryl of the complex is the rate-limiting step in the assay procedure.     

A multienzyme complex for CO2 fixation.

Acetyl-coenzyme A carboxylase from Euglena gracilis strain Z was isolated as a component of a multienzyme complex which includes phosphoenolpyruvate carboxylase and malate dehydrogenase. The multienzyme complex was shown to exist in crude extracts and was purified to a homogeneous protein with a molecular weight of 360,000 by gel filtration. The ratio of the activities of the constituent enzymes was acetyl-CoA carboxylase:phosphoenolpyruvate carboxylase:malate dehydrogenase, 1:25:500. The complex is proposed to operate in conjunction with malic enzyme, which is present in Euglena, to facilitate the formation of substrates, malonyl-CoA, and NADPH, for fatty acid biosynthesis. The interaction of the enzymes may represent a means of control of acetyl-CoA carboxylase activity in organisms which do not possess an enzyme subject to allosteric regulation. The acetyl-CoA carboxylase activity from Euglena is unaffected by citrate and isocitrate.     

Purification and characterization of fatty acid synthetase from Cryptococcus neoformans

Fatty acid synthetase has been purified from Cryptococcus neoformans 450 fold to a specific activity of 3.6 units per mg protein with an overall yield of 23%. The purified enzyme contained two non-identical subunits, Mr approximately 2.1×10⁵ and 1.8×10⁵. Under optimum conditions, 100 mM KCl and pH 7.5, apparent Km values for the substrates were: Acetyl CoA, 19 M; Malonyl CoA, 5 M; and NADPH, 6 M. Product inhibition patterns were determined to be: CoA, competitive versus acetyl CoA and malonyl CoA, uncompetitive versus NADPH; NADP, competitive versus NADPH, uncompetitive versus acetyl CoA and malonyl CoA; Palmitoyl CoA, competitive versus malonyl CoA, noncompetitive versus acetyl CoA and NADPH; Bicarbonate, uncompetitive versus malonyl CoA. These product inhibition patterns are consistent with the multisite ping-pong mechanism previously proposed for the avian fatty acid synthetase complex. The cryptococcal fatty acid synthetase was inhibited by the polyanionic polymers, heparin and dextran sulfate, an effect never before demonstrated for a fatty acid synthetase. This inhibition exhibited a marked dependence on the length of the polymer chain, with dextran sulfate fractions with Mr of 6×10⁵ and above having K _i values below 100 nanomolar. A model is presented that involves initial binding of the anionic polymer to the enzyme complex at a region of high positive charge density, followed by interaction of the end of the tethered polymer with the catalytic site. This study represents the first purification of fatty acid synthetase from a basidiomycete.     

Substrate inhibition of pigeon liver fatty acid synthetase and optimum assay conditions for over-all synthetase activity

The effects of the substrates acetyl-CoA, malonyl-CoA, and NADPH on the activity of pigeon liver fatty acid synthetase have been studied over a wide range of concentrations. Double-reciprocal coordinate plots for each of the substrates have been found to be linear at low concentrations. At higher concentrations two of the substrates, acetyl-CoA and malonyl-CoA, inhibit the rate of fatty acid synthesis. This double substrate inhibition is apparently of a competitive type. Inhibition by acetyl-CoA is very strong as compared to that by malonyl-CoA. At a 4:1 ratio of acetyl- to malonyl-CoA, inhibition is about 75%, whereas at a 4:1 ratio of malonyl- to acetyl-CoA fatty acid synthesis proceeds at the maximum rate.These results are consistent with the hypothesis that a competition between acetyl-CoA and malonyl-CoA occurs for the occupany of the 4′- phosphopantetheine site, a prosthetic group of the synthetase complex, and possibly also for the hydroxyl binding site (or sites). The relative concentrations of these substrates and the binding constants for each then determine whether these sites are occupied by acetyl or malonyl groups, and whether inhibition of fatty acid synthesis occurs. Based on our results, assays for pigeon liver fatty acid synthetase activity should be conducted at substrate concentrations of 15 μm, 60 μm, and 100 μm for acetyl-CoA, malonyl-CoA, and NADPH, respectively.     

Inhibition of mammalian fatty acid synthetase activity by NADP involves decreased mobility of the 4'-phosphopantetheine prosthetic group

Mammalian fatty acid synthetase carrying a 3-keto, 3-hydroxy, or 2-enoyl acyl-enzyme intermediate on the 4'-phosphopantetheine thiol is reversibly inhibited by binding of NADP to the enoyl reductase domain. Acyl moieties which can normally leave the enzyme by thioester hydrolysis or by transfer to a CoA acceptor cannot readily be removed from the NADP-inhibited enzyme; in addition, 3-keto or 2-enoyl moieties attached to the enzyme 4'-phosphopantetheine cannot readily be reduced when NADP is replaced by NADPH, even though model substrates can be reduced immediately. Reactivation of the NADP-inhibited 3-ketoacyl-enzyme, by exposure to NADPH, is paralleled by reduction and dehydration of the 3-ketoacyl moiety to a saturated acyl moiety without accumulation of either the 3-hydroxy or 2-enoyl acyl-enzyme intermediates, indicating that once the 4'-phosphopantetheine engages the ketoacyl moiety in the ketoreductase domain, subsequent reactions occur very rapidly. The results are consistent with a hypothesis which proposes that NADP binding to the enoyl reductase domain of fatty acid synthetase carrying an acyl intermediate other than a saturated moiety induces a conformational change in the enzyme that results in decreased mobility of the 4'-phosphopantetheine prosthetic group. Normal mobility of the prosthetic group, essential for transfer of acyl-enzyme intermediates through the active sites of the various functional domains, is restored relatively slowly when NADP is replaced by NADPH. It remains to be determined whether this modulation by pyridine nucleotides observed in vitro plays a role in the regulation of fatty acid synthetase activity in vivo.     

The purification and function of acetyl coenzyme A:acyl carrier protein transacylase

When individual enzyme activities of the fatty acid synthetase (FAS) system were assayed in extracts from five different plant tissues, acetyl-CoA:acyl carrier protein (ACP) transacylase and beta-ketoacyl-ACP synthetases I and II had consistently low specific activities in comparison with the other enzymes of the system. However, two of these extracts synthesized significant levels of medium chain fatty acids (rather than C16 and C18 acid) from [14C]malonyl-CoA; these extracts had elevated levels of acetyl-CoA:ACP transacylase. To explore the role of the acetyl transacylase more carefully, this enzyme was purified some 180-fold from spinach leaf extracts. Varying concentrations of the transacylase were then added either to spinach leaf extracts or to a completely reconstituted FAS system consisting of highly purified enzymes. The results suggested that: (a) acetyl-CoA:ACP transacylase was the enzyme catalyzing the rate-limiting step in the plant FAS system; (b) increasing concentration of this enzyme markedly increased the levels of the medium chain fatty acids, whereas increase of the other enzymes of the FAS system led to increased levels of stearic acid synthesis; and (c) beta-ketoacyl-ACP synthetase I was not involved in the rate-limiting step. It is suggested that modulation of the activity of acetyl-CoA:ACP transacylase may have important implications in the type of fatty acid synthesized, as well as the amount of fatty acids formed.     

Mammalian fatty acid synthetase. III. Characterization of human liver synthetase products and kinetics of methylmalonyl-CoA inhibition.

When propionyl-CoA was substituted for either acetyl-CoA or butyryl-CoA in the presence of [14C]malonyl-CoA and NADPH, the pure human liver fatty acids synthetase complex synthesized only straight-chain, saturated, 15- and 17-carbon radioactive fatty acids. At optimal concentrations, propionyl-CoA was a better primer of fatty acid synthesis than acetyl-CoA. Methylmalonyl-CoA inhibited the synthetase competitively with respect to malonyl-CoA. The Ki was calculated to be 8.4 muM. These findings provide an in vitro model and offer a direct explanation at the molecular level for some of the abnormal manifestations observed in diseases characterized by increased cellular concentrations of propionyl-CoA and methylmalonyl-CoA.     

Fatty acid synthetase from chloroplasts of soybean cotyledons: ACP activation and CoA inhibition

Fatty acid synthetase from chloroplasts of soybean cotyledons was activated by preincubation with acyl carrier protein and dithiothreitol. The synthetase reaction had a 3–10 min lag which was not eliminated by the preincubation. Acetyl-CoA and malonyl-CoA had no effect on the activation. Fatty acid synthetase from spinach chloroplasts was neither activated by preincubation nor had a lag. The variability of the activity of the soybean enzyme with preincubation suggested that the fatty acid synthetase was present in two forms and that the acyl carrier protein caused conversion to the active form. This fatty acid synthetase and the same synthetase from spinach chloroplasts were inhibited by CoA. The type of inhibition by CoA in soybean was competitive with respect to malonyl-CoA and the Ki was 80μM.     

Synthesis of fatty acids from malonyl-CoA and NADPH by pigeon liver fatty acid synthetase

Pigeon liver fatty acid synthetase has been found to catalyze the formation of palmitic acid from malonyl-CoA and NADPH in the absence of acetyl-CoA. Radio-chemical and spectral assays show that the activity of the complex in the absence of acetyl-CoA is about 25–30% of the activity in the presence of this compound. Initial velocities were determined for a series of reactions in which the malonyl-CoA concentration was varied over a range of 5–200 μm at a fixed NADPH concentration of 100μm and vice versa. No inhibitory effects of one substrate over the other were found. However, when the synthesis of fatty acids was studied in the presence of acetyl-CoA, a significant inhibitory effect of malonyl-CoA was observed. It has also been shown that the fatty acid synthetase synthesizes triacetic lactone from malonyl-CoA in the absence of NADPH and acetyl-CoA. No evidence was obtained for the direct decarboxylation of malonyl-CoA to acetyl-CoA in this reaction. Hence it is proposed that decarboxylation of the malonyl moiety bound covalently to 4′-phosphopantetheine occurs to yield acetyl-4′-phosphopantetheine. Further, it is proposed that the acetyl moiety of the latter compound is transferred to the cysteine site of the enzyme complex and that fatty acid synthesis proceeds in the presence of NADPH as proposed by Phillips et al. [Arch. Biochem. Biophys.138, 380 (1970)]. In the absence of NADPH triacetic lactone is formed.     

The physiological role of oxygen-sensitive pyruvate dehydrogenase in mitochondrial fatty acid synthesis in Euglena gracilis

In Euglena gracilis a malonyl-CoA-independent fatty acid-synthetic system, in which fatty acids are synthesized directly from acetyl-CoA as both primer and C2 donor, occurs in mitochondria, and the system contributes to the wax ester fermentation. The activity of fatty acid synthesis in the mitochondrial system was enhanced about six times when an artificial acetyl-CoA-regenerating system was present, indicating that the fatty acid-synthetic activity is controlled by the ratio of acetyl-CoA against CoA. When fatty acids were synthesized using pyruvate instead of acetyl-CoA as substrate, a high activity, about 30 times higher than that from acetyl-CoA, was found under anaerobic conditions (below 10(-5) M oxygen), while in aerobiosis fatty acids were not synthesized at all. CoA, NADH, and NADP+ were required as cofactors for fatty acid synthesis from pyruvate. It was indicated that high activity of fatty acid synthesis from pyruvate due to the high ratio of acetyl-CoA against CoA was maintained by the action of the oxygen-sensitive pyruvate dehydrogenase found in Euglena mitochondria. When [2-14C]pyruvate was fed into intact mitochondria under anaerobic conditions, radioactive fatty acids were formed in the presence of malate, which provided reducing power for the matrix.     

Regulation of plant Fatty Acid biosynthesis : analysis of acyl-coenzyme a and acyl-acyl carrier protein substrate pools in spinach and pea chloroplasts

In previous work (D. Post-Beittenmiller, J.G. Jaworski, J.B. Ohlrogge [1991] J Biol Chem 266: 1858-1865), the in vivo acyl-acyl carrier protein (ACP) pools were measured in spinach (Spinacia oleracea) leaves and changes in their levels were compared to changes in the rates of fatty acid biosynthesis. To further examine the pools of substrates and cofactors for fatty acid biosynthesis and to evaluate metabolic regulation of this pathway, we have now examined the coenzyme A (CoA) and short chain acyl-CoA pools, including acetyl- and malonyl-CoA, in isolated spinach and pea (Pisum sativum) chloroplasts. In addition, the relationships of the acetyl- and malonyl-CoA pools to the acetyl- and malonyl-ACP pools have been evaluated. These studies have led to the following conclusions: (a) Essentially all of the CoA (31-54 μm) in chloroplasts freshly isolated from light-grown spinach leaves or pea seedling was in the form of acetyl-CoA. (b) Chloroplasts contain at least 77% of the total leaf acetyl-CoA, based on comparison of acetyl-CoA levels in chloroplasts and total leaf. (c) CoA-SH was not detected either in freshly isolated chloroplasts or in incubated chloroplasts and is, therefore, less than 2 μm in the stroma. (d) The malonyl-CoA:ACP transacylase reaction is near equilibrium in both light- and dark-incubated chloroplasts, whereas the acetyl-CoA:ACP transacylase reaction is far from equilibrium in light-incubated chloroplasts. However, the acetyl-CoA:ACP transacylase reaction comes nearer to equilibrium when chloroplasts are incubated in the dark. (e) Malonyl-CoA and -ACP could be detected in isolated chloroplasts only during light incubations, and increased with increased rates of fatty acid biosynthesis. In contrast, both acetyl-CoA and acetyl-ACP were detectable in the absence of fatty acid biosynthesis, and acetyl-ACP decreased with increased rates of fatty acid biosynthesis. Together these data have provided direct in situ evidence that acetyl-CoA carboxylase plays a regulatory role in chloroplast fatty acid biosynthesis.     

Identity of malonyl and palmitoyl transferase of fatty acid synthetase from yeast. Functional interrelationships between the acyl transferases.

Functional interrelationships between the acyl transferases of yeast fatty acid synthetase were investigated. In binding assays with synthetase modified by 5,5'-dithiobis(2-nitrobenzoic acid), 4--5 malonyl transferase entities per multienzyme complex molecule could be titrated. In the presence of palmitoyl-CoA these malonyl transferases were found inaccessible to malonyl-CoA, whereas the acetyl transferases were reactive towards acetyl-CoA. Between four and five palmitoyl transferase entities per synthetase equivalent were found reactive towards palmitoyl-CoA, the palmitoyl binding being inhibited by malonyl-CoA. Following palmitoyl binding the acetyl transferases were found towards acetyl-CoA. Substrate model assays were consistent with these data. It is concluded that malonyl and palmitoyl transferases are closely coupled enzyme components of the multienzyme complex which are fairly independent of the acetyl transferase entities. The molecular basis for the observed coupling will be given in the following paper.     

Synthesis of long chain acyl-enzyme thioesters by modified fatty acid synthetases and their hydrolysis by a mammary gland thioesterase.

The fatty acid synthetase multienzyme from lactating rat mammary gland was modified either by removal of the two thioesterase I domains with trypsin or by inhibiting the thioesterase I activity with phenylmethanesulfonyl fluoride. The modified multienzymes are able to convert acetyl-CoA, malonyl-CoA, and NADPH to long chain acyl moieties (C₁₆C₂₂), which are covalently bound to the enzyme through thioester linkage, but they are unable to release the acyl groups as free fatty acids. A single enzyme-bound, long chain acyl thioester is formed by each molecule of modified multienzyme. Kinetic studies showed that the modified multienzymes rapidly elongate the acetyl primer moiety to a C₁₆ thioester and that further elongation to C₁₈, C₂₀, and C₂₂ is progressively slower. Thioesterase II, a mammary gland enzyme which is not part of the fatty acid synthetase multienzyme, can release the acyl moiety from its thioester linkage to either modified multienzyme. Kinetic data are consistent with the formation of an enzyme—substrate complex between thioesterase II and the acylated modified multienzymes. The present study demonstrates that the ability of thioesterase II to modify the product specificity of normal fatty acid synthetase is most likely attributable to the capacity of thioesterase II for hydrolysis of acyl moieties from thioester linkage to the multienzyme.     

Nature of the Fatty Acid Synthetase Systems in Parenchymal and Epidermal Cells of Allium porrum L. Leaves

Fatty acid synthesis was compared in cell-free extracts of epidermis and parenchyma of Allium porrum L. leaves. Parenchyma extracts had the major fatty acid synthetase (FAS) activity (70-90%) of the whole leaf; palmitic acid was also the major fatty acid synthesized when acetyl-coenzyme A (CoA) was the primer, but when acetyl-acyl carrier protein (ACP) was employed, C_18:0 and C_16:0 were synthesized in equal proportion. With the epidermal FAS system when either acetyl-CoA or acetyl-ACP was tested in the presence of labeled malonyl-CoA, palmitic acid was the only product synthesized. Specific activities of the FAS enzyme activities were determined in both tissue extracts.

The properties of malonyl-CoA:ACP transacylase were examined from the two different tissues. The molecular weights estimated by Sephadex G-200 chromatography were 38,000 for the epidermal enzyme and 45,000 for parenchymal enzyme. The optimal pH was for both enzymes 7.8 to 8.0 and the maximal velocity 0.4 to 0.5 micromoles per milligram protein per minute. These enzymes had different affinities for malonyl-CoA and ACP. For the malonyl-CoA:ACP transacylase of epidermis, the K_m values were 5.6 and 13.7 micromolar for malonyl-CoA and ACP, respectively, and 4.2 and 21.7 micromolar for the parenchymal enzyme. These results suggest that the FAS system in both tissues are nonassociated, that the malonyl-CoA:ACP transacylases are isozymes, and that both in epidermis and in parenchyma tissue two independent FAS system occur. Evidence would suggest that β-ketoacyl-ACP synthase II is present in the parenchymal cells but missing in the epidermal cell.

     

Presence of one essential arginine that specifically binds the 2′-phosphate of NADPH on each of the ketoacyl reductase and enoyl reductase active sites of fatty acid synthetase

Two arginine modifying reagents, phenylglyoxal and 2,3-butanedione, inactivated fatty acid synthetase from goose uropygial gland. This inactivation could be partially prevented by NADP, 2′-AMP, and 2′,5′-ADP, whereas acetyl-CoA and/or malonyl-CoA provided very little protection. Ketoacyl reductase and enoyl reductase activities of fatty acid synthetase showed similar inactivation by phenylglyoxal and butanedione and protection by only NADP and its 2′-phosphate-containing analogs. Furthermore, 2′-AMP was found to be a competitive inhibitor of overall fatty acid synthetase, ketoacyl reductase, and enoyl reductase with apparent K_i values of 1.4, 0.2, and 14 mm, respectively. These results suggest that binding of NADPH to fatty acid synthetase involves specific interaction of the 2′-phosphate with the guanidino group of arginine residues at the active site of the two reductases. Quantitation of the number of arginine residues modified revealed that 4 out of 106 arginine residues per subunit of the synthetase showed high reactivity toward phenylglyoxal. Scatchard analysis showed that two rapidly reacting arginine residues had no effect on the catalytic activity, while modification of two additional arginine residues resulted in complete loss of enzyme activity. Under these conditions, of the seven partial reactions of fatty acid synthetase, only the ketoacyl reductase and enoyl reductase activities were inhibited by phenylglyoxal. The differential reversal of inhibition of the two reductases and the overall activity of fatty acid synthetase, resulting from dialysis of the modified enzyme, suggested that both ketoacyl reductase sites and enoyl reductase sites are required for the full expression of fatty acid synthetase activity. The results of the present chemical modification studies are consistent with the hypothesis that each subunit of fatty acid synthetase contains one ketoacyl reductase and one enoyl reductase and suggest that one essential arginine is present at each of these active sites.     

Overproduction of a Functional Fatty Acid Biosynthetic Enzyme Blocks Fatty Acid Synthesis in Escherichia coli

β-Ketoacyl-acyl carrier protein (ACP) synthetase II (KAS II) is one of three Escherichia coli isozymes that catalyze the elongation of growing fatty acid chains by condensation of acyl-ACP with malonyl-ACP. Overexpression of this enzyme has been found to be extremely toxic to E. coli, much more so than overproduction of either of the other KAS isozymes, KAS I or KAS III. The immediate effect of KAS II overproduction is the cessation of phospholipid synthesis, and this inhibition is specifically due to the blockage of fatty acid synthesis. To determine the cause of this inhibition, we examined the intracellular pools of ACP, coenzyme A (CoA), and their acyl thioesters. Although no significant changes were detected in the acyl-ACP pools, the CoA pools were dramatically altered by KAS II overproduction. Malonyl-CoA increased to about 40% of the total cellular CoA pool upon KAS II overproduction from a steady-state level of around 0.5% in the absence of KAS II overproduction. This finding indicated that the conversion of malonyl-CoA to fatty acids had been blocked and could be explained if either the conversion of malonyl-CoA to malonyl-ACP and/or the elongation reactions of fatty acid synthesis had been blocked. Overproduction of malonyl-CoA:ACP transacylase, the enzyme catalyzing the conversion of malonyl-CoA to malonyl-ACP, partially relieved the toxicity of KAS II overproduction, consistent with a model in which high levels of KAS II blocks access of the other KAS isozymes to malonyl-CoA:ACP transacylase.     

Subunits of fatty acid synthetase complexes. Enzymatic activities and properties of the half-molecular weight nonidentical subunits of pigeon liver fatty acid synthetase.

The separation of the half-molecular weight, nonidentical subunits (I and II) of the pigeon liver fatty acid synthetase complex has been achieved on a large (20 mg) scale by affinity chromatography on Sepharose epsilon-aminocaproyl pantetheine. This separation requires a careful control of temperature, ionic strength, pH, and column flow rate for success. The yield of subunit II is further improved by transacetylation (with acetyl-CoA) of the dissociated fatty acid synthetase prior to affinity chromatography. The separated subunit I (reductase) contains the 4'-phosphopantetheine (A2) acyl binding site, two NADPH binding sites, and beta-ketoacyl and crotonyl thioester reductases. Subunit II (transacylase) contains the B1 (hydroxyl or loading) and B2 (cysteine) acyl binding sites, and acetyl- and malonyl-CoA: pantetheine transacylases. When subunit I is mixed in equimolar quantities with subunit II, an additional NADPH binding site is found even though subunit II alone shows no NADPH binding. Both subunits contain activities for the partial reactions, beta-hydroxybutyryl thioester dehydrase (crotonase) and palmityl-CoA deacylase. Subunit I has 8 sulfhydryl groups per mol whereas subunit II has 60. Reconstitution of fatty acid synthetase activity to 75% of the control level is achieved on reassociation of subunits I and II.     

Inhibition in vitro of lipogenic enzymes from bovine (Bos taurus) mammary tissue by methylmalonyl-coenzyme A and coenzyme A

Bovine mammary fatty acid synthetase was inhibited by approximately 50% by 40 microM methylmalonyl-CoA; this inhibition was competitive with respect to malonyl-CoA (apparent Ki = 11 microM). Similarly, 6.25 microM coenzyme A inhibited the synthetase by 35% and this inhibition was again competitive (apparent Ki = 1.7 microM). Apparent Km for malonyl-CoA was 29 microM. The short-chain dicarboxylic acids malonic, methylmalonic and ethylmalonic at high concentrations (160-320 microM) and ATP (5 mM) enhanced the synthetase activity by about 50% respectively; the activating effects of methylmalonic acid and ATP on the synthetase were additive. Methylmalonyl-CoA at 50 microM concentration inhibited the partially purified acetyl-CoA carboxylase uncompetitively by 10% and the propionyl-CoA carboxylase activity of the enzyme preparation competitively (apparent Ki = 21 microM) by 40%. Malonyl-CoA also inhibited the acetyl-CoA carboxylase activity competitively (apparent Ki = 7 microM) by 35% and the propionyl-CoA carboxylating activity of the preparation competitively (apparent Ki = 4 microM) by 82%. The possibility that methylmalonyl-CoA may be a causal factor in the aetiology of the low milk-fat syndrome in high yielding dairy cows is discussed.