Carboxylation Of acetyl-CoA Research Articles

Acetyl Coenzyme A Carboxylase: IV. BIOTINYL PROSTHETIC GROUP-INDEPENDENT MALONYL COENZYME A DECARBOXYLATION AND CARBOXYL TRANSFER: GENERALIZATION TO OTHER BIOTIN ENZYMES

Abstract Liver acetyl-CoA carboxylase, a biotin-enzyme which catalyzes the ATP-dependent carboxylation of acetyl-CoA (acceptor) to form malonyl-CoA (carboxylated acceptor), decarboxylates malonyl-CoA by a biotin-dependent, as well as a biotin-independent mechanism. Neither ADP, Pi, nor divalent metal ion are required for either of these abortive decarboxylations. The biotin-dependent reaction is blocked by avidin and is activated synergistically by acetyl-CoA and citrate, an allosteric effector of the carboxylase. However, the biotin-independent process is unaffected by avidin and citrate, and is inhibited slightly by acetyl-CoA. The occurrence of this abortive reaction indicates that the enzyme promotes the labilization of the free carboxyl group of malonyl-CoA and that insertion of a proton in its place is possible without the participation of the biotinyl prosthetic group. This phenomenon appears to be a property of biotin-dependent carboxylases since pig heart propionyl-CoA carboxylase, the biotin-free carboxyltransferase component of Escherichia coli acetyl-CoA carboxylase, and avian liver pyruvate carboxylase catalyze similar biotin-independent decarboxylations of their respective carboxylated acceptor substrates. Liver acetyl-CoA carboxylase also catalyzes carboxyl transfer from [3-14C]malonyl-CoA to free d-biotin, a model for the second half-reaction in the over-all carboxylation process. The acid-labile radioactive carboxylation product was stabilized by methylation with diazomethane, and the ester formed identified as 1'-N-carboxy-d-biotin dimethyl ester by carrier recrystallization with the authentic compound. The model reaction does not involve the covalently bound biotinyl prosthetic group, since the essentially irreversible complex formation of this group with avidin does not block carboxyl transfer. While the reaction is specific in that l-biotin and imidazolidone-2 are inactive, certain closely related biotin derivatives including d-homobiotin and biocytin (e-N-d-biotinyl-l-lysine) are somewhat better acceptor substrates than d-biotin. The fact that citrate blocks carboxyl transfer to free d-biotin and biocytin with both avidin-complexed and native carboxylase without affecting the rate of malonyl-CoA decarboxylation is compatible with earlier evidence implicating the biotinyl prosthetic group as the focal point of citrate-induced conformational changes.

Comparative effects of dietary regimens on the levels of enzymes regulating the synthesis of fatty acids and cholesterol in rat liver

The activities, in subcellular fractions of rat liver, for acetyl-CoA carboxylation, fatty acid synthesis, and β-hydroxy-β-methylglutaryl-CoA reduction and for the overall conversion of acetate to cholesterol were determined for animals in different nutritional states. Acetyl-CoA carboxylase and fatty acid synthetase activities increased or decreased together whenever the nutritional state was changed. A similar relationship was observed between β-hydroxy-β-methylglutaryl-CoA reductase activity and the synthesis of cholesterol from acetate. Feeding a fat-free diet after either a normal or fasted regimen increased the activity of acetyl-CoA carboxylase and fatty acid synthetase but suppressed the activities for β-hydroxy-β-methylglutaryl CoA reduction and cholesterol synthesis. Thus, the quantity of fat in the diet apparently regulates the levels of activity of the key enzymes of two divergent pathways in an opposite manner. The levels of these enzymes in turn control the proportion of acetyl-CoA used by each pathway. When a diet containing 2% cholesterol is fed, the levels of β-hydroxy-β-methylglutaryl-CoA reductase activity and cholesterol synthesis in liver are markedly depressed. In contrast, the levels of acetyl-CoA carboxylase and fatty acid synthetase activities are unaffected. These results suggest that cholesterol and fat exert their effects on the cholesterol-synthesizing and fatty acid-synthesizing pathways by different mechanisms.

Fatty acid biosynthesis: IX. Fatty acid synthesis from enzymically and chemically acetylated rabbit mammary gland fatty acid synthetase

1. I. [I- 14C]Acetate was covalently bound to rabbit mammary gland fatty acid synthetase by enzymic transacylation from [I- 14C]acetyl-CoA. Per mole of enzyme 2 moles of acetate were bound to thiol groups and up to I mole of acetate was bound to non-thiol groups. 2. 2. The acetyl-fatty acid synthetase complex was isolated free from acetyl-CoA. It was rapidly hydrolysed at 30°C, but hydrolysis was greatly diminished at o°C and triacetic lactone synthesis occurred. In the presence of malonyl-CoA and NADPH, all the acetate bound to fatty acid synthetase was incorporated into long-chain fatty acids. Hydrolysis of bound acetate and incorporation of bound acetate into fatty acids were inhibited to the same extent by guanidine hydrochloride. 3. 3. Acetate was also covalently bound to fatty acid synthetase by chemical acetylation with [I- 14C]acetic anhydride in the absence of CoASH. A total of 60 moles of acetate were bound per mole of fatty acid synthetase, with 20–25 moles being bound to thiol groups per mole of enzyme. Acylation did not inhibit enzyme activity. The majority of the bound acetate was stable to hydrolysis at o °C. Out of the 60 moles of acetate bound per mole of acetylated enzyme, up to 20 moles were incorporated into fatty acids in the presence of malonyl-CoA and NADPH. 4. 4. Due to the rapid hydrolysis of acetyl-fatty acid synthetase prepared from both acetyl-CoA and acetic anhydride, direct carboxylation of acetyl-fatty acid synthetase to form malonyl-fatty acid synthetase could not be demonstrated by CO 2 fixation under the experimental conditions used. With acetic anhydride as acetyl donor, there was a low rate of incorporation of acetate into fatty acids in the presence of NADPH but absence of added malonyl-CoA. This could be due to direct carboxylation of the acetate bound to fatty acid synthetase or, if any CoA were associated with the fatty acid synthetase, to carboxylation of acetyl-CoA formed by chemical acetylation of this CoA.

Fatty acid biosynthesis. VIII. The fate of malonyl-CoA in fatty acid biosynthesis by purified enzymes from lactating-rabbit mammary gland

1. 1. We have investigated the formation and utilization of malonyl-CoA in fatty acid synthesis catalysed by preparations of partially purified acetyl-CoA carboxylase and purified fatty acid synthetase from lactating-rabbit mammary gland. 2. 2. Carboxylation of [1- 14C]acetyl-CoA was linked to fatty acid synthesis by the presence of fatty acid synthetase and NADPH. The rate of fatty acid formation was equal to that of acetyl-CoA carboxylation, without the accumulation of free malonyl-CoA to a concentration required to obtain the same rate of fatty acid synthesis from added [1,3- 14C 2]malonyl-CoA. 3. 3. The preparations of acetyl-CoA carboxylase and fatty acid synthetase were each able to decarboxylate [1,3- 14C 2]malonyl-CoA. 4. 4. Both enzyme preparations acted as competitive inhibitors of 14CO 2 fixation into acetyl-CoA catalysed by acetyl-CoA carboxylase in the absence of NADPH. This is attributed to a mechanism of product inhibition. The effect on the apparent activity of acetyl-CoA carboxylase assayed by malonyl-CoA formation is discussed. 5. 5. Our results suggest a metabolic compartmentation of the carboxylation step which facilitates the incorporation of carboxylated acetyl-CoA into fatty acids in the presence of NADPH and prevents its catabolism by side reactions.

The carboxyl transferase component of acetyl CoA carboxylase: structural evidence for intersubunit translocation of the biotin prosthetic group.

An essential protein component of acetyl CoA carboxylase, isolated and extensively purified from cell-free extracts of Escherichia coli, has been identified as malonyl CoA: d-biotin carboxyl transferase. This enzyme, which does not contain covalently-bound biotin, catalyzes carboxyl transfer from malonyl CoA to free d-biotin, a model reaction for the second step in the carboxylation of acetyl CoA. The transcarboxylation product, after stabilization by methylation, was identified as 1'-N-carboxy-d-biotin dimethyl ester. These results indicate the presence of a biotin site on the carboxyl transferase, distinct from that on the biotin carboxylase, which carries out the first step in the overall process. In addition, the carboxyl transferase catalyzes a slower abortive decarboxylation of malonyl CoA, thus indicating that carboxyl abstraction and protonation do not require the participation of biotin. It is now evident that the half-reactions of acetyl CoA carboxylation are catalyzed by biotin carboxylase and carboxyl transferase. Both components are devoid of biotin and have specific binding sites for free d-biotin, as well as for their respective substrates; hence, the acetyl CoA carboxylation mechanism must involve intersubunit translocation of the carboxylated biotinyl group, which is bound covalently to carboxyl-carrier-protein, a noncatalytic polypeptide.

Tricarboxylic Acid Activator-Induced Changes at the Active Site of Acetyl-CoA Carboxylase

Publisher Summary This chapter focuses on tricarboxylic acid activator-induced changes at the active site of acetyl-CoA carboxylase. Acetyl-CoA carboxylase catalyzes the first committed step of extramitochondrial fatty acid synthesis in animal tissues and appears to be an important control point in this biosynthetic pathway. The carboxylase appears to be regulated both by alterations of its level in tissues and by modification of its catalytic efficiency with allosteric effectors. Certain tricarboxylic acids, notably citrate and isocitrate, markedly activate the carboxylase-catalyzed reaction. Activation by these acids is rather specific in that tricarballylate, a close structural analog, is completely inactive. Citrate appears to serve the dual role in fatty acid synthesis of precursor of extramitochondrial acetyl-CoA via the citrate-cleavage reaction and of “feed-forward” activator of acetyl-CoA carboxylase. Activation by tricarboxylic acids is accomplished by the rapid polymerization of carboxylase protomers to give rise to polymeric filaments. In this chapter, effect of activator on the partial reactions of acetyl-CoA carboxylation is analyzed. Effect of activator on the environment of the prosthetic group is elaborated. Effect of activator on the reactivity of the carboxylated biotin prosthetic group is also described.

Acetyl CoA carboxylase, II. Deomonstration of biotin-protein and biotin carboxylase subunits.

Previous work has shown that Escherichia coli acetyl CoA carboxylase is composed of two dissimilar protein components, E(a) which contains covalently bound biotin and forms E(a)-CO(2)-from HCO(3)- and ATP, and E(b) which is involved in the transfer of the carboxyl group from E(a)-CO(2)- to acetyl CoA, forming malonyl CoA. E(a) has been dissociated into two subunits at pH 9. One subunit, designated biotin carboxylase, catalyzes a model reaction, the ATP-dependent carboxylation of free (+)-biotin. The other subunit contains covalently bound which is carboxylated by the biotin carboxylase in the course of acetyl CoA carboxylation.

Liver Acetyl Coenzyme A Carboxylase: I. ISOLATION AND CATALYTIC PROPERTIES

Abstract Acetyl coenzyme A carboxylase has been isolated from chicken liver and purified more than 1000-fold in good yield. It is an unusually stable protein exhibiting negligible loss of enzymatic activity during 1 week of storage (1 to 5 mg of protein per ml) at room temperature in potassium phosphate buffer, pH 7. The pure enzyme catalyzes the carboxylation of 8.8 µmoles of acetyl-CoA (or 7.0 µmoles of propionyl-CoA) per min per mg of refractometrically determined protein at its pH optimum of 7.5 at 37°. The carboxylase-catalyzed carboxylation of acetyl-CoA is activated 15- to 16-fold by dl-isocitrate or citrate and 5-fold by malonate; tricarballylate is essentially inactive. Arrhenius plots for acetyl-CoA carboxylation in the presence or absence of isocitrate are biphasic, having a transition point at about 21°. Below this temperature, where there is very little activator effect, the temperature coefficient (Q10) in the presence of isocitrate is 8.0. Above the transition point, where isocitrate activation is evident, the temperature coefficient (Q10) in the presence of activator is 2.0 and in its absence is a negative value. At pH 7.5 and 37°, the carboxylase catalyzes the over-all reverse reaction (decarboxylation), ATP-32Pi exchange, malonyl-CoA-14C-acetyl-CoA exchange, and ADP-, Pi-, and Mg2+-independent malonyl-CoA decarboxylation reactions at 14%, 6.2%, 33%, and 1.2%, respectively, of the rate of the over-all forward (carboxylation) reaction. All of the above mentioned reactions exhibit essentially the same tri- and dicarboxylic acid activation patterns. Evidence is presented which indicates that isocitrate activation of the carboxylase is associated with an increased maximum velocity.

Lipid synthesis in Lipomyces lipofer.

The synthesis of lipids by cell-free fractions of Lipomyces lipofer was investigated by measuring the incorporation of acetate-14C and other radioactive precursors into lipid-soluble material. On the basis of cofactor requirements, the reaction to inhibitors, and the ability to incorporate appreciable quantities of malonate-14C, it was concluded that the malonyl-CoA pathway was the principal route of fatty acid synthesis in this yeast. By employing a means of cell dispersion less rigorous than previously used with yeasts, it was shown that the soluble portion (high-speed supernatant) of the cell was primarily involved in lipid synthesis. The mitochondrial fraction and the high-speed pellet (96 000 × g, 1 h) were inactive although the latter fraction was required for the maximum activity of the high-speed supernatant. The stimulatory activity of this pellet was very labile and showed a lag period before it became effective. Several lines of evidence suggested that the carboxylation of acetyl-CoA is the rate-limiting step in lipid synthesis which is accelerated by the high-speed pellet.

Liver acetyl CoA carboxylase: insight into the mechanism of activation by tricarboxylic acids and acetyl CoA.

Recent investigations in this laboratory have shownl, 2 that the isocitrate(or citrate-) activated form of liver acetyl CoA carboxylase (E.C. 6.4.1.2) is a large protein structure having a molecular weight of about four million. Electron microscopic examination of the carboxylase in the presence of isocitrate reveals' that it has a filamentous structure with dimensions of 80-100 A by up to 5000 A. Reversible dissociation to inactive protomeric subunits (molecular weight 409,000) occurs2 at low enzyme concentration in the carboxylation assay reaction mixture in the absence of isocitrate or at higher enzyme concentration in the presence of 0.5 M NaCl at pH 8.0. Reconstitution to the active filamentous form can be accomplished either by addition of isocitrate (or citrate) or by removal of NaCl and introduction of isocitrate by dialysis. The carboxylation of acetyl CoA catalyzed by liver acetyl CoA carboxylase involves the following two partial reactions (reactions 1 and 2):

Regulation of fatty acid synthesis at the acetyl-CoA carboxylation step

The carboxylation of acetyl-CoA to form malonybCoA is the first and ratelimiting step in the biosynthetic sequence leading specifically to long-chain fatty acids. Studies in greater & tall on acetyl-CoA carboxylase which catalyzes this reaction are therefore of importance in relation to the regulation of fatty acid synthesis. With the acetyl-CoA carboxylase purified from rat liver we obtained the following results. 1. The active carboxyl in carboxylated acetyl-CoA carboxylase is bound, as in other carboxy-biofin-enzymes, to the l'-N-atom of biotin, which is amide-linked to the ~-amino group of a lysine residue in the enzyme protein. 2. Enzyme activated by citrate at 25°C is reversibly inactivated by exposure to cold (0 to 4° C). Sucrose gradient centrifugafion studies in medium containing citrate indicate that the sedimentation coefficient of the enzyme protein is larger at 25°C than at 4°C. 3. Enzyme is inhibited strongly by long-chain aeyl-CoA derivatives. The inhibition is competitive with regard to citrate and non-competitive with regard to aeetyl-CoA, bicarbonate or ATP. It was found by sucrose gradient centrifugation studies that the enzyme sediments more slowly in the presence of palmityl-CoA than in its absence. A possible significance of the in vitro findings in the cellular regulation of fatty acid synthesis is discussed. Inhibition of acetyl-CoA carboxylase by long-chain acyl-CoA derivatives might represent a negative feedback mechanism.

FORMATION OF ETHYL ACETATE IN FERMENTATION WITH BREWER'S YEAST: IV. METABOLISM OF ACETYL-COENZYME A

During fermentation with Saccharomyces cerevisiae, esters are formed from alcohols and acyl-CoA. The formation of ethyl acetate thus depends on the availability of acetyl-CoA, which is formed partly by activating acetic acid (requiring ATP) and partly by oxidative decarboxylation of pyruvic acid (requiring lipoic acid and thiamine pyrophosphate participation). Formation of ethyl acetate is stimulated by addition of small quantities of DNP (2,4-dinitrophenol), an effect which is enhanced by addition of lipoic acid and counteracted by sodium arsenite (an inhibitor to lipoic acid). These results show that oxidative decarboxylation of pyruvic acid is an important contributive factor to anaerobic formation of acetyl-CoA by Sacch. cerevisiae. Formation of ethyl butyrate from butyric acid is inhibited by DNP in all concentrations; butyrate must first be activated to butyryl-CoA (requiring ATP). Formation of ethyl acetate is inhibited by higher concentrations of DNP, owing to diminished oxidation to acetate in different states, to inhibition of formation of coenzyme A from pantothenic acid (requiring ATP) or to the enzymatic pattern of the cells becoming changed; all these factors may play some part. Formation of esters is stimulated when other reactions requiring acetyl-CoA are inhibited, e.g., the citric acid cycle and carboxylation of acetyl-CoA to malonyl-CoA, which can be achieved by making the medium biotin-free. Malonic acid is also an inhibitor of the citric acid cycle and interacts in lipid synthesis.

Fatty acid synthesis by complex systems The possibility of regulation by microsomes

1. 1. Fatty acid synthesis from malonyl-CoA catalyzed by rat-liver microsomal protein does not seem to be due to contamination with enzymes from the particle-free supernatant or mitochondrial fractions. 2. 2. Optimum conditions for the conversion of malonyl-CoA to fatty acids by rat-liver microsomes with respect to substrate concentration, protein concentration, and incubation time were established. The enzyme systems concerned with fatty acid synthesis from malonyl-CoA in the particle-free supernatant fraction were 4–5 times more active than those in the microsomes. 3. 3. Preincubation of the microsomes caused a release of malonyl-CoA decarboxylase (EC 4.1.1.9) but not of fatty acid synthetase. This resulted in a greater conversion of the available malonyl-CoA to fatty acids. 4. 4. The products of synthesis in the supernatant system were free fatty acids bound to protein, whereas the products of the microsomal system were predominantly complex lipids (phospholipid). 5. 5. The predominant fatty acid synthesized from malonyl-CoA by the supernatant fraction was palmitate, and that synthesized by the microsomes was stearate. 6. 6. In the system composed of the supernatant fraction plus microsomes, the fatty acids synthesized were complex lipids (phospholipids), and the pattern of fatty acid synthesis resembled that found in the liver slice. 7. 7. The mechanism of the stimulatory effect of microsomes on fatty acid synthesis by the supernatant fraction is discussed and tentatively localized at the level of acetyl-CoA carboxylation. The stimulation may be due to a release of feedback inhibition.

Fatty acid synthesis by complex systems The possibility of regulation by microsomes

1. 1. Fatty acid synthesis from malonyl-CoA catalyzed by rat-liver microsomal protein does not seem to be due to contamination with enzymes from the particle-free supernatant or mitochondrial fractions. 2. 2. Optimum conditions for the conversion of malonyl-CoA to fatty acids by rat-liver microsomes with respect to substrate concentration, protein concentration, and incubation time were established. The enzyme systems concerned with fatty acid synthesis from malonyl-CoA in the particle-free supernatant fraction were 4–5 times more active than those in the microsomes. 3. 3. Preincubation of the microsomes caused a release of malonyl-CoA decarboxylase (EC 4.1.1.9) but not of fatty acid synthetase. This resulted in a greater conversion of the available malonyl-CoA to fatty acids. 4. 4. The products of synthesis in the supernatant system were free fatty acids bound to protein, whereas the products of the microsomal system were predominantly complex lipids (phospholipid). 5. 5. The predominant fatty acid synthesized from malonyl-CoA by the supernatant fraction was palmitate, and that synthesized by the microsomes was stearate. 6. 6. In the system composed of the supernatant fraction plus microsomes, the fatty acids synthesized were complex lipids (phospholipids), and the pattern of fatty acid synthesis resembled that found in the liver slice. 7. 7. The mechanism of the stimulatory effect of microsomes on fatty acid synthesis by the supernatant fraction is discussed and tentatively localized at the level of acetyl-CoA carboxylation. The stimulation may be due to a release of feedback inhibition.

Enzymic carboxylation of butyryl coenzyme A. I. Conversion of butyryl coenzyme A to 2-ethylmalonyl coenzyme A

1. 1. The presence of an enzyme in dog skeletal and heart muscle and ox, chicken, and rat liver, which catalyzes the fixation of C 14O 2 by butyryl coenzyme A (CoA) is described. The reaction requires adenosine triphosphate (ATP) and Mn ++ or Mg ++. 2. 2. The relative rates of carboxylation of acetyl-CoA, propionyl-CoA, and butyryl-CoA in these tissues is about 1:25:3, suggesting that one enzyme—propionyl-CoA carboxylase—is involved in all three reactions. In rat liver mitochondria the rates of carboxylation of propionyl-CoA and butyryl-CoA are almost equal. 3. 3. The product of carboxylation of butyryl-CoA has been identified as 2-ethyl-malonyl-CoA. Reversibility of the carboxylation reaction was demonstrated with synthetic 2-ethylmalonyl-CoA. 4. 4. Rat liver mitochondrial extract catalyzes the synthesis of 2-ethylmalonyl-CoA from 2-ethylmalonate, adenosine triphosphate and coenzyme A. 5. 5. Methylmalonyl-CoA isomerase preparations from kidney and Propionibacterium shermanii do not isomerize 2-ethylmalonyl-CoA to either glutaryl-CoA or methylsuccinyl-CoA. 6. 6. The significance of the butyryl-CoA carboxylation reaction and the metabolism of 2-ethylmalonyl-CoA are discussed.

Biosynthesis of Fatty Acids: I. STUDIES WITH ENZYMES OBTAINED FROM LIVER

The requirement for bicarbonate in the incubation medium for the conversion of octanoic acid to stearic acid by slices of liver tissue was reported some 10 years ago (1). Furthermore, the inclusion of bicarbonate in the homogenizing medium permitted the preparation of a consistently active enzyme system from pigeon liver which catalyzed the conversion of acetate or acetyl coenzyme A to long chain fatty acids (2). The significance of these observations was not fully realized until the complete dependence of fatty acid synthesis on the presence of HCO,-, adenosine 5’-triphosphate, and Mg++ or Mn++ (3, 4), in addition to acetyl coenzyme A, was demonstrated. The requirement for CO% for fatty acid synthesis was also demonstrated in an enzyme system obtained from avocado (5), intact yeast (6)) and cell-free preparations of yeast (7). The finding that HCQwas not incorporated into long chain fatty acids by enzyme systems which require the presence of COZ (3, 4) suggested that acetyl-CoA was first carboxylated to malonyl-CoA. Accordingly, malonyl-CoA was synthesized and was found to be a required precursor for the synthesis of long chain fatty acids in an enzyme system obtained from pigeon liver tissue (8). The enzymatic carboxylation of acetyl-CoA was demonstrated soon thereafter (7, 9, lo), and the participation of this reactant in fatty acid synthesis was fully confirmed (11-13). The original demonstrations of the conversion of malonyl-CoA to long chain fatty acids (8) were performed with crude enzyme preparations obtained from pigeon liver which catalyzed the reduction of acetyl-CoA by TPNH to acetaldehyde. This finding led to the tentative proposal that elongation of the carbon chain of fatty acids might occur by reduction of acyl-CoA derivatives to the respective aldehydes, followed by condensation between the carbonyl carbon of the aldehyde with the methylene carbon of malonyl-CoA (8). Experiments with purified enzyme preparations indicate that such a mechanism is probably incorrect. It is the purpose of the present communications to report the results obtained with purified enzyme preparations which afford a clearer insight into the nature and the sequence of reactions that occur in the biosynthesis of long chain fatty acids.

Influence of previous diet on hepatic glycogenesis and lipogenesis.

Influence of previous diet on hepatic glycogenesis and lipogenesis.