Regulation of fatty acid synthesis.

Acetyl-CoA carboxylase and fatty acid synthetase are the two major enzymes involved in the synthesis of fatty acids in animals. The activities of both enzymes are affected by nutritional manipulations. Although acetyl-CoA carboxylase is considered generally to be the rate-limiting step in lipogenesis, there is evidence that suggests that fatty acid synthetase may become rate limiting under certain conditions. The principal support for the view that acetyl-CoA carboxylase is the rate-limiting enzyme for lipogenesis is that the activity of the enzyme is controlled by allosteric effectors that change the catalytic efficiency of the enzyme. Until recently, the only known control of fatty acid synthetase was through changes in rate of enzyme synthesis. Data are reviewed that show that fatty acid synthetase can exist in forms possessing different catalytic activities. Thus fatty acid synthetase appears to be subject to the type of control necessary for an enzyme to serve as a regulator of the rate of a biological process over a short term.     

Mechanism of fatty acid synthesis

Significant advances have been made in the past few years in our understanding of the mechanism of synthesis of fatty acids, the structural organization of fatty acid synthetase complexes and the mechanism of regulation of activity of these enzyme systems. Numerous fatty acid synthetase complexes have been purified to homogeneity and the mechanism of synthesis of fatty acids by these enzyme systems has been ascertained from tracer, and recently, kinetic studies. The results obtained by these methods are in complete agreement. Furthermore, the kinetic results have indicated that fatty acid synthesis proceeds by a seven-site ping-pong mechanism. Several of the fatty acid synthetases have been dissociated completely to nonidentical half-molecular weight subunit species and these have been separated by affinity chromatography. From one of these subunits acyl carrier protein has been obtained. Whether the nonidentical subunits can be dissociated into individual proteins or whether these subunits are each comprised of one peptide is still a matter of controversy. However, it appears to us that each of the half-molecular weight subunits is indeed comprised of individual proteins. Studies on the regulation of activity of fatty acid synthetase complexes of avian and mammalian liver have resulted in the separation by affinity chromatography of three species (apo, holo-$\text{a}$ and holo-$\text{b}$) of fatty acid synthetase. Since these species have radically different enzyme activities they may provide a mechanism of short-term regulation of fatty acid synthetase activity. Other studies have shown that the quantity of avian and mammalian liver fatty acid synthetases is controlled by a change in the rate of synthesis of this enzyme complex. This change in the rate of synthesis of enzyme complex is under the control of insulin and glucagon. The former hormone increases the rate of enzyme synthesis, whereas the latter decreases it. Further studies on fatty acid synthetase complexes will undoubtedly concentrate upon more refined aspects of the structural organization of these enzyme systems, including the sequencing of acyl carrier proteins, the effects of protein-protein interaction on the kinetics of the partial reactions of fatty acid synthesis catalyzed by separated enzymes of the complex, the mechanism of hormonal regulation of fatty acid synthetase activity and x-ray diffraction analysis of subunits and complex.     

Medium-chain fatty acid synthesis by goat mammary-gland fatty acid synthetase. The effect of limited proteolysis.

Fatty acid synthetase from goat mammary gland was subjected to limited proteolysis by trypsin and elastase. Both proteolytic enzymes selectively cleaved the chain-terminating thioester hydrolase component from the enzyme complex, leaving all other partial activities intact in the core peptides. Trypsin, but not elastase, caused extensive degradation of the released thioester hydrolase. The released thioester hydrolase could be purified to homogeneity by gel filtration. The molecular weight was estimated as 29 000 and the enzyme showed only significant hydrolytic activity toward long-chain acyl-CoA esters. The core peptides retained the ability to synthesize medium-chain acyl-CoA esters in the presence of 2,6-di-O-methyl-alpha-cyclodextrin. The results conclusively show that the terminating thioester hydrolase of goat mammary-gland fatty acid synthetase is not involved in termination of medium-chain-length fatty acid synthesis by this enzyme.     

Cell-free synthesis of pigeon liver fatty acid synthetase.

Pigeon liver fatty acid synthetase proteins (apo- and holo-forms) have been synthesized in a cell-free system reconstituted from polysomes and a soluble enzyme fraction. Identification of the cell-free synthesized products as fatty acid synthetase was achieved by affinity chromatography, by immuno-precipitation and by the simultaneous conversion of both the authentic carrier protein and the $\text{in vitro}$ synthesized products from the holo- to the apo-form of the synthetase. The reverse conversion was also effected.     

Inhibition of plant fatty acid synthesis by nitroimidazoles.

1. The effect of the addition of a number of nitroimidazoles was tested on fatty acid synthesis by germinating pea seeds, isolated lettuce chloroplasts and a soluble fraction from pea seeds. 2. All the compounds tested had a marked inhibition on stearate desaturation by lettuce chloroplasts and on the synthesis of very-long-chain fatty acids by pea seeds. 3. In contrast, the effect of the drugs on total fatty acid synthesis from [14C]acetate in chloroplasts was related to the compound's electron reduction potentials. 4. Of the compounds used, only metronidazole had a marked inhibition on palmitate elongation in the systems tested. 5. The mechanism of inhibition of plant fatty acid synthesis by nitroimidazoles is discussed and the possible relevance of these findings to their neurotoxicity is suggested.     

Fatty acid synthesis by spinach chloroplasts I. Property of fatty acid synthesis from acetate

Intact chloroplasts (about 70% Class I chloroplasts) isolatedfrom spinach leaves incorporated 150 nmoles of [1-¹⁴C] acetateinto fatty acids per mg chlorophyll in 1 hr at pH 8.3, 25°Cand 25,000 lux. On electron and phase-contrast microscopiescombined with hypotonic treatment of chloroplasts, this syntheticactivity was shown to be proportional to the percentage of ClassI chloroplasts in the preparation. Light was necessary for thesynthesis, the activity in the complete reaction mixture inthe dark being only 2% of that in the light. The synthetic activityincreased with increasing intensities of light to reach saturationat 6,000 lux. CoA and ATP were most effective as cofactors,HCO₃^–, HPO₄^2–, Mg^2$ and Mn^2$ were less effective.ATP could be replaced by ADP in the presence of Pi, suggestingpossible supply of ATP by photophosphorylation. Omission ofthe NADPH-generation system and NADH did not affect the synthesis,indicating sufficient provision of endogenous NADPH and NADHin intact chloroplasts under light. Addition of DTE did notcause recovery of the synthetic activity of intact chloroplastsin the dark. ¹ Present address: Radioisotope Centre, University of Tokyo,Yayoi, Bunkyo, Tokyo 113, Japan. (Received August 26, 1974; )     

Mercury inhibition of fatty acid synthesis in chicks.

Male chicks were fed a commercial ration and were given drinking water which contained 0, 50, 100, 150, 200 or 300 mug of mercury/ml as mercuric chloride from hatching to 3 weeks of age. In one experiment, the mercuric chloride was administered by injection into the abdominal cavity rather than in the drinking water. At 3 weeks the chicks were killed, and the livers were removed and weighed. The activity of fatty acid synthetase in the 800 X gav supernatant fractions of the liver homogenates and in vivo incorporation of [14C]acetate into liver and carcass fatty acids and respiratory 14CO2 was determined as indicated. Administration of mercury at a treatment level of 300 mug/ml of drinking water depressed growth, feed and water consumption, liver weight, hepatic fatty acid synthetase activity, and in vivo incorporation of [14C]acetate into liver and carcass fatty acids, and increased the production of respiratory 14CO2 as compared with controls. In experiments in which graded doses of mercury were administered, body weights, liver weights, and feed and water intakes of the chicks receiving 0, 50 and 100 mug of mercury/ml of drinking water were similar to each other, but these parameters were severely depressed by 200 mug of mercury/ml of drinking water. Mercury caused a dose-related decrease of fatty acid synthetase activity. Incorporation of [14C]acetate into carcass fatty acid was depressed by 50 and 200 mug of mercury/ml of drinking water; incorporation into liver fatty acids and production of respiratory 14CO2 was not affected by mercury. Intra-abdominal injection of 6 mg of mercury/100 g body weight (as mercuric chloride) into well alimented chicks depressed hepatic fatty acid synthetase activity at 1 h post-injection. The data are consistent with the hypothesis that a portion of the effects of mercury on fatty acid synthesis are direct rather than a secondary effect of inanition.     

Light control of fatty acid synthesis and diurnal fluctuations of fatty acid composition in leaves.

1. Although isolated spinach chloroplasts were almost entirely (greater than 99%) dependent on light for fatty acid synthesis, leaf discs were capable of fatty acid synthesis in the dark (up to 500nmol of 3H/h per mg of chlorophyll equivalent to approx. 400nmol of carbon/h per mg of chlorophyll), which represented 12-20% of the corresponding 'light rates'. 2. Net fatty acid accumulation by greening maize leaves occurred largely or entirely during the light period. 3. There was a diurnal fluctuation in the proportions of C18 unsaturated fatty acids in the lipids of developing spinach leaves, where an increase in the concentration of oleate during the day and a subsequent decline at night was observed; a complementary change occurred in the concentration of alpha-linolenate. The rhythm is interpreted as reflecting the continuation of oleate and linoleate desaturation at high rates when oleate synthesis is markedly decreased at night. 4. Changes in the fatty acid composition of 3-sn-phosphatidylcholine accounted for at least 60% of the total decrease in oleate over the dark period. This result is consistent with suggestions that this lipid is the substrate for the leaf microsomal oleate desaturase and an intermediate in leaf glycerolipid biosynthesis.     

Inhibiting bacterial fatty acid synthesis

The type II fatty acid synthase consists of a series of individual enzymes, each encoded by a separate gene, that catalyze discrete steps in chain elongation. The formation of fatty acids is vital to bacteria, and each of the essential enzymes and their acyl group carriers represent a potential target for the development of novel antibacterial therapeutics. High resolution x-ray and/or NMR structures of representative members of every enzyme in the type II pathway are now available, and these structures are a valuable resource to guide antibacterial drug discovery. The role of each enzyme in regulating pathway activity and the diversity in the components of the pathway in the major human pathogens are important considerations in deciding the most suitable targets for future drug development.     

Carbon flux and fatty acid synthesis in plants.

The de novo synthesis of fatty acids in plants occurs in the plastids through the activity of fatty acid synthetase. The synthesis of the malonyl-coenzyme A that is required for acyl-chain elongation requires the import of metabolites from the cytosol and their subsequent metabolism. Early studies had implicated acetate as the carbon source for plastidial fatty acid synthesis but more recent experiments have provided data that argue against this. A range of cytosolic metabolites including glucose 6-phosphate, malate, phosphoenolpyruvate and pyruvate support high rates of fatty acid synthesis by isolated plastids, the relative utilisation of which depends upon the plant species and the organ from which the plastids are isolated. The import of these metabolites occurs via specific transporters on the plastid envelope and recent advances in the understanding of the role of these transporters are discussed. Chloroplasts are able to generate the reducing power and ATP required for fatty acid synthesis by capture of light energy in the reactions of photosynthetic electron transport. Regulation of chloroplast fatty acid synthesis is mediated by the response of acetyl-CoA carboxylase to the redox state of the plastid, which ensures that the carbon metabolism is linked to the energy status. The regulation of fatty acid synthesis in plastids of heterotrophic cells is much less well understood and is of particular interest in the tissues that accumulate large amounts of the storage oil, triacylglycerol. In these heterotrophic cells the plastids import ATP and oxidise imported carbon sources to produce the required reducing power. The sequencing of the genome of Arabidopsis thaliana has now enabled a number of aspects of plant fatty acid synthesis to be re-addressed, particularly those areas in which in vitro biochemical analysis had provided equivocal answers. Examples of such aspects and future opportunities for our understanding of plant fatty acid synthesis are presented and discussed.     

Initiation of fatty acid synthesis in rat mammary glands.

The rate of fatty acid synthesis from [6-14C]glucose in mammary tissue remained low until parturition at 22 days of gestation and increased 10-fold at 1 day post partum. Administration of progesterone on days 20 and 21 or removal of pups at parturition abolished this increase. In the latter case, administration of prolactin, corticosterone or oxytocin had no stimulatory effect; tissue from suckled glands in which the ducts had been ligated at parturition also showed no increased rate in 24 h. Foetoplacentectomy on day 18 did not stimulate fatty acid synthesis but subsequent suckling by foster pups did. Whereas lactose synthesis is initiated by withdrawal of progesterone from the circulation, a further stimulus related to removal of milk by suckling is required to initiate fatty acid synthesis.     

Fatty acid synthesis by spinach chloroplasts III. Relationship between fatty acid synthesis and photophosphorylation

The modes of actions of photosynthetic inhibitors on photosynthesisand fatty acid synthesis were examined. DCMU, an electron transport inhibitor, inhibited fatty acidsynthesis and photophosphorylation to the same extent, suggestingdependence of fatty acid synthesis on photosynthesis. The samewas also the case with FCCP, a photophosphorylation uncoupler.In contrast, NH₄Cl and phlorizin at concentrations completelysuppressing ATP formation, only partially inhibited the fattyacid synthesis. These facts suggest that a certain level ofhigh-energy intermediate (state) is responsible for the lightenhancement of fatty acid synthesis. This idea is further supportedby the fact that the partial inhibition of fatty acid synthesisby NH₄Cl was relieved by addition of DCCD at low concentrationssuppressing the ATP formation but not completely destroyingthe high energy intermediate. The lag period in the initial period of fatty acid synthesiswas shortened by preillumination of chloroplasts, even in theabsence of ADP. This indicates that the light dependent fattyacid synthesis is closely associated with the high-energy intermediate(state), but not directly with ATP formation by photophosphorylation. ¹ Present address: Radioisotope Centre, University of Tokyo,Yayoi, Bunkyo, Tokyo 113, Japan. (Received August 26, 1974; )