Decarboxylation Of malonyl-CoA Research Articles

Dissection of malonyl-coenzyme A decarboxylation from polyketide formation in the reaction mechanism of a plant polyketide synthase.

Chalcone synthase (CHS) catalyzes formation of the phenylpropanoid chalcone from one p-coumaroyl-CoA and three malonyl-coenzyme A (CoA) thioesters. The three-dimensional structure of CHS [Ferrer, J.-L., Jez, J. M., Bowman, M. E., Dixon, R. A., and Noel, J. P. (1999) Nat. Struct. Biol. 6, 775-784] suggests that four residues (Cys164, Phe215, His303, and Asn336) participate in the multiple decarboxylation and condensation reactions catalyzed by this enzyme. Here, we functionally characterize 16 point mutants of these residues for chalcone production, malonyl-CoA decarboxylation, and the ability to bind CoA and acetyl-CoA. Our results confirm Cys164's role as the active-site nucleophile in polyketide formation and elucidate the importance of His303 and Asn336 in the malonyl-CoA decarboxylation reaction. We suggest that Phe215 may help orient substrates at the active site during elongation of the polyketide intermediate. To better understand the structure-function relationships in some of these mutants, we also determined the crystal structures of the CHS C164A, H303Q, and N336A mutants refined to 1.69, 2.0, and 2.15 A resolution, respectively. The structure of the C164A mutant reveals that the proposed oxyanion hole formed by His303 and Asn336 remains undisturbed, allowing this mutant to catalyze malonyl-CoA decarboxylation without chalcone formation. The structures of the H303Q and N336A mutants support the importance of His303 and Asn336 in polarizing the thioester carbonyl of malonyl-CoA during the decarboxylation reaction. In addition, both of these residues may also participate in stabilizing the tetrahedral transition state during polyketide elongation. Conservation of the catalytic functions of the active-site residues may occur across a wide variety of condensing enzymes, including other polyketide and fatty acid synthases.

Conversion of a beta-ketoacyl synthase to a malonyl decarboxylase by replacement of the active-site cysteine with glutamine.

beta-Ketoacyl synthases involved in the biosynthesis of fatty acids and polyketides exhibit extensive sequence similarity and share a common reaction mechanism, in which the carbanion participating in the condensation reaction is generated by decarboxylation of a malonyl or methylmalonyl moiety; normally, the decarboxylation step does not take place readily unless an acyl moiety is positioned on the active-site cysteine residue in readiness for the ensuing condensation reaction. Replacement of the cysteine nucleophile (Cys-161) with glutamine, in the beta-ketoacyl synthase domain of the multifunctional animal fatty acid synthase, completely inhibits the condensation reaction but increases the uncoupled rate of malonyl decarboxylation by more than 2 orders of magnitude. On the other hand, replacement with Ser, Ala, Asn, Gly, and Thr compromises the condensation reaction without having any marked effect on the decarboxylation reaction. The affinity of the beta-ketoacyl synthase for malonyl moieties, in the absence of acetyl moieties, is significantly increased in the Cys161Gln mutant compared to that in the wild type and is similar to that exhibited by the wild-type beta-ketoacyl synthase in the presence of an acetyl primer. These results, together with modeling studies of the Cys --> Gln mutant from the crystal structure of the Escherichia coli beta-ketoacyl synthase II enzyme, suggest that the side chain carbonyl group of the Gln-161 can mimic the carbonyl of the acyl moiety in the acyl-enzyme intermediate so that the mutant adopts a conformation analogous to that of the acyl-enzyme intermediate. Catalysis of the decarboxylation of malonyl-CoA requires the dimeric form of the Cys161Gln fatty acid synthase and involves prior transfer of the malonyl moiety from the CoA ester to the acyl carrier protein domain and subsequent release of the acetyl product by transfer back to a CoA acceptor. These results suggest that the role of the Cys --> Gln beta-ketoacyl synthases found in the loading domains of some modular polyketide synthases likely is to act as malonyl, or methylmalonyl, decarboxylases that provide a source of primer for the chain extension reactions catalyzed by associated modules containing fully competent beta-ketoacyl synthases.

Plant polyketide synthases leading to stilbenoids have a domain catalyzing malonyl-CoA:CO2 exchange, malonyl-CoA decarboxylation, and covalent enzyme modification and a site for chain lengthening.

Stilbene synthases and the related bibenzyl synthases are plant polyketide synthases whose biological functions lie in the formation of antimicrobial phytoalexins. The formation of hydroxystilbenes from one molecule of acyl-CoA and three molecules of malonyl-CoA is catalyzed by a homodimeric 90 kDa protein and includes Claisen condensations and cleavage of a thioester followed by decarboxylation. Combining inhibitor studies, protein modifications, and site-directed mutagenesis, we were able to differentiate between the binding sites for malonyl-CoA and the regions responsible for the selection of the primer, p-coumaroyl-CoA or m-hydroxyphenylpropionyl-CoA, respectively. Mutations in the C-terminal part of the molecule or modification by photolabeling with p-azidocinnamoyl-CoA influence the overall reaction, the formation of hydroxystilbenes, but leave partial reactions, such as the malonyl-CoA:CO2 exchange and the malonyl-CoA-dependent modification of the enzyme, unaffected. Data obtained with several kinds of stilbene synthase and mutant forms suggest that the malonyl-CoA-dependent covalent modification takes place at a cysteine residue in the N-terminal part of the enzyme. Mutations in the C-terminal half of the enzyme molecule do not interfere with the malonyl-CoA-dependent reactions.

Anaerobic malonate decarboxylation by Citrobacter diversus. Growth and metabolic studies, and evidence of ATP formation.

Citrobacter diversus ATCC 27156 was able to grow by decarboxylation of malonate to acetate under strictly anaerobic conditions, in the presence of yeast extract. The growth yield, corrected for growth on yeast extract, was 2.03 g cell dry mass per mol malonate. The addition of malonate to ATP-depleted cell suspensions (less than 0.2 nmol ATP/mg cell protein) resulted in a rapid increase in cellular ATP levels to between 4.5 and 6.0 nmol/mg cell protein. Intact cells decarboxylated malonate at rates of up to 1.5 mumol/min.mg protein. Enzyme assays on malonate-grown cells indicated activation of malonate by an ATP-dependent ligase reaction and by CoA transfer from acetyl-CoA, followed by decarboxylation of malonyl-CoA to acetyl-CoA with subsequent recovery of the invested ATP by substrate level phosphorylation through the activity of acetate kinase. Net ATP synthesis is postulated to be mediated by gradient formation coupled to the decarboxylation of malonyl-CoA. The protonophore CCCP and H(+)-ATPase inhibitor DCCD significantly reduced cellular ATP levels, suggesting a role for proton gradients in the energy metabolism of this strain when growing an malonate. Inhibitors of sodium metabolism or ommission of sodium had no effect on ATP levels or malonate decarboxylation.

On the mechanism of sodium ion translocation by methylmalonyl‐CoA decarboxylase from Veillonella alcalescens

Veillonella alcalescens during lactate degradation developed an Na+ concentration gradient with 7-8 times higher external than internal Na+ concentrations in the logarithmic growth phase. The gradient declined to a factor of 1.9 in the late stationary phase. Methylmalonyl-CoA decarboxylase reconstituted into proteoliposomes performed an active electrogenic Na+ transport, creating delta psi of 60 mV, delta pNa+ of 50 mV, and delta mu Na+ of 110 mV. In the initial phase of the transport, the decarboxylase catalyzed the uptake of 2 Na+ ions malonyl-CoA molecule decarboxylated. During further development of the electrochemical Na+ gradient, this ratio gradually declined to zero, when decarboxylation continued without further increase of the internal Na+ concentration. The rate of malonyl-CoA decarboxylation declined initially during development of the membrane potential, but remained unchanged later on. Monensin abolished the Na+ gradient and increased the malonyl-CoA decarboxylation rate 2.8-fold. On dissipating the membrane potential with valinomycin, the internal Na+ concentration reached three times higher values than in its absence, and the decarboxylation rate increased 2.8-fold. Methylmalonyl-CoA decarboxylase catalyzed an exchange of internal and external Na+ ions in addition to net Na+ accumulation. The initial rate of Na+ influx was double that of malonyl-CoA decarboxylation. In the following, both rates decreased about twofold in parallel to values which remained constant during further development of the electrochemical Na+ gradient. Thus, Na+ influx and malonyl-CoA decarboxylation follow a stoichiometry of approximately 2:1, independent of the magnitude of the electrochemical Na+ gradient and are thus highly coupled events.

Decarboxylation of malonyl-CoA by lactating bovine mammary fatty acid synthase

1. 1. A pronounced malonyl-CoA decarboxylase activity of bovine mammary fatty acid synthase results in the formation of acetyl-CoA and not of triacetic acid lactone as in the reaction by yeast and pigeon liver synthase. 2. 2. This activity is unaffected by the dissociation of the enzyme and is insensitive to its modification by iodoacetamide, N-ethylmaleimide, p-hydroxymercuribenzoate or 2-chloroacetyl-CoA. 3. 3. A 50% inhibition of the activity observed on the depletion of free CoA from the medium indicates that at least part of the reaction occurs only after the acylation of the enzyme with the malonyl group. 4. 4. A parallel reaction without such a transfer also appears to occur simultaneously.

Mechanism of activation of bovine kidney pyruvate dehydrogenase a kinase by malonyl-CoA and enzyme-catalyzed decarboxylation of malonyl-CoA.

The activity of the pyruvate dehydrogenase kinase, which phosphorylates and thereby inactivates the pyruvate dehydrogenase complex, was stimulated by malonyl-CoA. Treatment with [2-14C]malonyl-CoA resulted in acylation of sites in the complex. Both acylation and activation of kinase activity increased in a time-dependent manner with a parallel increase in those activities when the malonyl-CoA:CoA ratio was varied. Protein-bound acyl groups were labilized by performic acid treatment indicating their attachment to protein at thiol residues; however, the product released was volatile, which is not characteristic of malonic acid. While malonyl-CoA was initially free of acetyl-CoA, stimulation of kinase activity and acylation of sites in the complex by malonyl-CoA were shown to be contingent upon enzyme-catalyzed decarboxylation. Decarboxylation appeared to be catalyzed by a trace contaminant present in highly purified preparations of both the pyruvate and 2-oxoglutarate dehydrogenase complexes. Under conditions in which both free CoA was removed (by conversion to succinyl-CoA) and then, after various periods, free acetyl-CoA was removed (by enzymic conversion to acetyl phosphate), both acetylation of sites in the complex and activation of kinase activity increased in a time-dependent manner. Concomitantly there was a decrease in the concentration dependence for activation of the kinase by malonyl-CoA. Our results strongly support the conclusion that activation of kinase activity is associated with acylation of sites in the complex, and that, in the case of malonyl-CoA, those processes depend on enzyme-catalyzed decarboxylation.

Assay of fatty acid synthetase by mass fragmentography using [13C]malonyl-CoA.

A new assay method for fatty acid synthetase using mass fragmentography was described. [2-13C]Malonyl-CoA was chemically synthesized from [2-13C]malonic acid and used as a substrate. The newly synthesized fatty acids were quantitated with a GC-MS instrument after methyl esterification. Monitoring of molecular ions of the newly synthesized fatty acids enabled us to determine the absolute amounts with heptadecanoic acid as an internal standard. Multiple products (14 : 0, 16 : 0, and 18 : 0) were measured individually. Using this technique, we obtained information about production profiles such as that of chain length against incubation temperature and about malonyl-CoA decarboxylation activity in enzyme preparations, and we also confirmed the presence of malonyl-CoA decarboxylation activity even in purified fatty acid synthetase from guinea pig Harderian gland. Compared with the conventional assay methods (spectrophotometric and radioisotopic), this method was more reliable and useful.

Malonyl-CoA Decarboxylase from Streptomyces erythreus: Purification, properties, and possible role in the production of erythromycin

Malonyl-CoA decarboxylase was purified (800-fold) from an erythromycin-producing strain of Streptomyces erythreus using DEAE-cellulose, Sephadex G-100, SP-Sephadex, and gel filtration with Sephadex G-75. The molecular weight of the native enzyme was 93,000 as determined by gel filtration and the subunit molecular weight was 45,000 as estimated by sodium dodecyl sulfate-polyacrylamide electrophoresis, suggesting an α 2 subunit composition for the native enzyme. Evidence is presented that during the purification procedure and storage a proteolytic cleavage occurred resulting in the formation of 30- and 15-kDa peptides. The enzyme showed a pH optimum of about 5.0 whereas the vertebrate enzyme showed an optimum at alkaline pH. The enzyme decarboxylated malonyl-CoA with a K m of 143 μ m and V of 250 nmol min −1 mg −1. For the decarboxylation of methylmalonyl-CoA this enzyme showed the opposite stereospecificity to that shown by vertebrate enzyme; the ( R) isomer was decarboxylated at 3% of the rate observed with malonyl-CoA while the ( S) isomer was not a substrate. Neither avidin nor biotin affected the rate of malonyl-CoA decarboxylation, suggesting that biotin is not involved in catalysis. Acetyl-CoA and free CoA were found to be competitive inhibitors. Propionyl-CoA, butyryl-CoA, succinyl-CoA, and methylmalonyl-CoA showed little inhibition, and neither thiol-directed reagents nor chelating agents inhibited the enzyme. High ionic strength and sulfate ions caused reversible inhibition of the enzymatic activity. Under two different cultural conditions the time course of appearance of malonyl-CoA decarboxylase was determined by measuring the enzyme activity and the level of the enzyme protein by an immunological method using rabbit antibodies prepared against the enzyme. In both cases the increase and decrease in the decarboxylase correlated with the rate of production of erythromycin, suggesting a possible role for this enzyme in the antibiotic production.

Propionyl-Coa induced synthesis of even-chain-length fatty acids by fatty acid synthetase from Brevibacterium ammoniagenes.

The product distribution of Brevibacterium ammoniagenes fatty acid synthetase has been investigated using propionyl-CoA instead of acetyl-CoA as the primer. The synthetase produces not only an odd-numbered fatty acid (heptadecanoic acid) but also even-numbered fatty acids (stearic and oleic acids) in the presence of propionyl-CoA. The amounts of heptadecanoic, stearic and oleic acids increased with increasing concentration of propionyl-CoA. However, the formation of heptadecenoic acid (C17:1) was not observed under any conditions tested. The failure of C17:1 synthesis suggested that the enzyme component catalyzing the beta, gamma-dehydration, which is responsible for the synthesis of unsaturated fatty acids, has a high degree of chain length specificity. Under standard assay conditions, stearic acid predominated and heptadecanoic and oleic acids were found in lesser amounts. Mass spectrometric analyses of fatty acids synthesized either from [2H]propionyl-CoA or in 2H2O revealed that propionyl-CoA is utilized as the priming substrate for the synthesis of heptadecanoic acid and that an acetyl residues, which is formed by the decarboxylation of malonyl-CoA, served as the priming substrate for the syntheses of stearic and oleic acids. No evidence was obtained for the direct decarboxylation of malonyl-CoA to acetyl-CoA in this reaction. It is concluded that the decarboxylation of the malonyl moiety bound to the synthetase occurs efficiently only in the course of fatty acid synthesis. A hypothetical scheme is presented to explain the propionyl-CoA-dependent decarboxylation of the malonyl moiety.

Kinetic analysis of the malonyl coenzyme A decarboxylation and the condensation reaction of fatty acid synthesis. Application to the study of malonyl coenzyme A inactivated chicken liver fatty acid synthetase.

A kinetic analysis of the decarboxylation of malonyl-CoA and the condensation--CO2 exchange reaction of fatty acid synthesis has been carried out. The analysis supported by experimental evidence defines conditions under which the decarboxylation of malonyl-CoA quantitatively reflects the activity for the condensation reaction between enzyme-bound acyl and malonyl groups. NADP+ decreases the release of 14CO2 from radiolabeled malonyl-CoA by lowering the rates of the processes leading to the formation of triacetic acid lactone. For accurate measurements, the enzyme concentration should be less than 200 micrograms/mL, and malonyl-CoA/enzyme ratios should be 200 or less. Short reaction periods (1 min or less) and inclusion of NADP+ (100 microM) enhance the accuracy of measurements. These analyses have been used to explain the mechanism of malonyl-CoA mediated inactivation of chicken liver fatty acid synthetase and are appropriate for determining the functional condensing site of the polyfunctional polypeptide chains comprising the dimeric enzyme.

Inactivation of chicken liver fatty acid synthetase by malonyl coenzyme A. Effects of acetyl coenzyme A and nicotinamide adenine dinucleotide phosphate

Chicken liver fatty acid synthetase complex is irreversibly inactivated by one of the substrates, malonyl-CoA. Acetyl-CoA has a dual role. At concentrations less than or comparable to those of malonyl-CoA, the rate of inactivation is enhanced, whereas at acetyl-CoA/malonyl-CoA ratios greater than 2, the rte of inactivation is slowed down. NADP+ at low concentrations (25 microM) affords considerable protection against malonyl-CoA mediated inactivation whereas NAD+ even at 1.0 mM concentration has no effect. The inactivation process does not lead to the dissociation of the enzyme complex and is accompanied by subtle conformational changes as measured by circular dichroism measurements. Of all the model partial reactions, decarboxylation of malonyl-CoA and the condensation--CO2 exchange are the only reactions which are not catalyzed by the modified species. The process of inactivation is accompanied by enhanced covalent binding of malonyl groups such that approximately 6 mol of the acyl group is bound per mol of the enzyme at complete inactivation. The available evidence suggests that the inactivation of the enzyme results from the binding of malonyl group(s) at or near the condensing site of the enzyme.

Flavanone synthase catalyzes CO 2 exchange and decarboxylation of malonyl-CoA

Flavanone synthase catalyzes CO 2 exchange and decarboxylation of malonyl-CoA

Malonyl-CoA decarboxylase from the uropygial gland of waterfowl: Purification, properties, immunological comparison, and role in regulating the synthesis of multimethyl-branched fatty acids

Analysis of the acyl portion of the wax from the uropygial gland of muscovy duck, wood duck, (Cairininae subfamily) and Canadian goose (Anserinae) by combined gas-liquid chromatography and mass spectrometry showed that 2,4,6-trimethyloctanoic acid and 2,4,6-trimethylnonanoic acid were the major (~100%) components. Similar analyses of the wax from the glands of mallard and Peking duck (Anatinae) showed that 2- and 4-mono-methylhexanoic acids predominated (>75%) with no multimethyl-branched acids. The uropygial glands of the former group contained 20 to 100 times as much malonyl-CoA decarboxylase activity as those of the latter group. These results strongly support the hypothesis that this decarboxylase, by causing specific decarboxylation of malonyl-CoA, makes available only methylmalonyl-CoA for fatty acid synthesis, and thus causes the production of multimethyl-branched acids. Malonyl-CoA decarboxylase was purified to apparent homogeniety in 30% yield from the uropygial glands of muscovy and wood ducks. Properties of the enzyme from the ducks, such as S 20.w (7.8 S), molecular weight (190,000) subunit composition (4 × 47,000), amino acid composition, strict substrate specificity, pH optimum (~9.0), K m (~33 μ m), V (~80 μmol/min/mg), and inhibition by SH-directed reagents were similar to those observed with the decarboxylase from the domestic goose. Antiserum prepared against the goose enzyme cross-reacted with and inhibited the decarboxylase from the four genera of ducks and Canadian goose. Ouchterlony double-diffusion analyses showed fusion of precipitant lines with the enzyme from muscovy, wood duck, and Canadian goose, whereas spurs were observed with the enzymes from mallard and Peking ducks. Immunoelectrophoresis showed that the decarboxylases from muscovy and wood ducks were similar and that they were different from the enzyme from the domestic goose. It appears that during evolution, the subfamilies (Anserinae and Cairininae) which synthesize multimethyl-branched acids acquired the ability to produce a high level of malonyl-CoA decarboxylase, an enzyme which is also present in low levels in other organisms.

Developmental changes in malonate-related enzymes of rat brain

Mitochondria and high-speed supernatant were prepared from rat brain homogenates at 0–50 days of age. The development of malonyl-CoA synthetase, malonyl-CoA decarboxylase, coenzyme A-transferases and acetyl-CoA hydrolase was examined and compared to de novo fatty acid biosynthesis. The specific activity of malonyl-CoA synthetase rose steeply between 6 and 10 days, and this sudden increase coincided with peak specific activity of fatty acid synthetase. Similarly, malonate activation by coenzyme A-transfer from succinyl-CoA increased rapidly at the same time. Transfer of the coenzyme A moiety from acetoacetyl-CoA was only minimal during this period. Brain mitochondria had active malonyl-CoA decarboxylase which showed an almost linear increase of specific activity between 0 and 50 days. Acetyl-CoA resulting from malonyl-CoA decarboxylation underwent enzymatic hydrolysis to acetate and free coenzyme A. Only traces of acetoacetate were recovered. In mitochondria, acetyl-CoA hydrolase increased progressively whereas the cytosolic enzyme had high specific activity at birth which declined slowly during maturation.

Lipid biosynthesis in sebaceous glands. Regulation of the synthesis of n- and branched fatty acids by malonyl-coenzyme A decarboxylase

Crude cell-free extracts isolated from the uropygial glands of goose catalyzed the carboxylation of propionyl-CoA but not acetyl-CoA. However, a partially purified preparation catalyzed the carboxylation of both substrates and the characteristics of this carboxylase were similar to those reported for chicken liver carboxylase. The Km and Vmax for the carboxylation of either acetyl-CoA or propionyl-CoA were 1.5 times 10- minus-5 M and 0.8 mumol per min per mg, respectively. In the crude extracts an inhibitor of the acetyl-CoA carboxylase activity was detected. The inhibitor was partially purified and identified as a protein that catalyzed the rapid decarboxylation of malonyl-CoA. This enzyme was avidin-insenitive and highly specific for malonyl-CoA with very low rates of decarboxylation for methylmalonyl-CoA and malonic acid. Vmax and Km for malonyl-CoA decarboxylation, at the pH optimum of 9.5, were 12.5 mumol per min per mg and 8 times 10- minus-4 M, respectively. The relative activities of the acetyl-CoA carboxylase and malonyl-CoA decarboxylase were about 4 mumol per min per gland and 70 mumoles per min per gland, respectively. Therefore acetyl-CoA and methylmalonyl-CoA should be the major primer and elongating agent, respectively, present in the gland. The major fatty acid formed from these precursors by the fatty acid synthetase of the gland would be 2,4,6,8-tetramethyl-decanoic acid which is known to be the major fatty acid of the gland (Buckner, J. S. and Kolattukudy, P. E. (1975), Biochemistry, following paper). Therefore it is concluded that the malonyl-CoA decarboxylase controls fatty acid synthesis in this gland.

Acetyl Coenzyme A Carboxylase System of Escherichia coli: STUDIES ON THE MECHANISMS OF THE BIOTIN CARBOXYLASE- AND CARBOXYLTRANSFERASE-CATALYZED REACTIONS

Abstract Stoichiometric, isotopic exchange and model reaction studies carried out with the resolved, homogeneous protein components (biotin carboxylase, carboxyltransferase, and biotin-containing carboxyl carrier protein) of the acetyl-CoA carboxylase system from Escherichia coli B show that the half-reactions of acetyl-CoA carboxylation occur on different subunits according to the following sequence: CCP (carboxyl carrier protein)-Biotin + HO-CO2- + ATP (Me2+)/⇌/(BC) CCP-biotin-CO2- + ADP + Pi (1) CCP-biotin-CO2- + acetyl-CoA ⇌/(CT) CCP-biotin + malonyl-CoA (2) Net: acetyl-CoA + HO-CO2- + ADP (Me2+)/⇌ malonyl-CoA + ADP + Pi (3) Biotin carboxylase catalyzes the stoichiometric carboxylation of carboxyl carrier protein in the presence of ATP, Mg2+, and HCO3-; ATP-[14C]ADP exchange; and ATP-32Pi exchange in accordance with Reaction 1; divalent cation, bicarbonate, and carboxyl carrier protein are required for both exchanges. Furthermore, ATP-[14C]ADP exchange requires inorganic phosphate, whereas ATP-32Pi exchange requires ADP. Carboxyltransferase carries out net transcarboxylation from the carboxylated carboxyl carrier protein to acetyl-CoA to form malonyl-CoA (Reaction 2) and in addition, catalyzes carboxyl carrier protein-dependent malonyl CoA-[14C]acetyl-CoA exchange. All of the exchange reactions are inhibited by avidin. The two catalytic components of the E. coli carboxylase system (biotin carboxylase and carboxyltransferase), although devoid of covalently bound biotin, catalyze model reactions with free d-biotin derivatives which account for their respective roles in Reactions 1 and 2 and show that each component possesses a specific binding site for the bicyclic ring of the prosthetic group. This is consistent with the view that the biotinyl prosthetic group acts as a mobile carboxyl carrier which oscillates between the carboxylation and transcarboxylation sites on biotin carboxylase and carboxyltransferase, respectively. The fact that biotin carboxylase also catalyzes rapid biotin-dependent phosphoryl transfer from carbamyl phosphate (an analogue of carbonic-phosphoric anhydride) to ADP to form ATP suggests that the carboxylation of free d-biotin or carboxyl carrier protein may proceed via a carbonic-phosphoric anhydride intermediate. In the phosphoryl transfer reaction, biotin appears to fulfill a conformational requirement of the enzyme rather than to participate in the reaction per se. Derivatives of biotin in which the site of carboxylation, i.e. the 1'-ureido-N (1'-N) is substituted and, therefore, inactive in the carboxylation reaction, support the phosphoryl transfer reaction. Moreover, the Km of biotin carboxylase for free d-biotin is more than two orders of magnitude lower for phosphoryl transfer (1 mm) than for carboxylation (170 mm). Carboxyltransferase, which catalyzes transcarboxylation from malonyl-CoA to free d-biotin or its derivatives, also catalyzes a slow decarboxylation of malonyl-CoA to form acetyl-CoA in the absence of a model carboxyl acceptor. 2-Imidazolidone, an analogue of the ureido ring of biotin, does not serve as an acceptor in the carboxyltransfer reaction, but accelerates malonyl-CoA decarboxylation. Thus, although the transferase per se activates C—C bond cleavage in malonyl-CoA, this process is enhanced by binding the 2-imidazolidone ring at the transcarboxylation site. All of the model reactions exhibit similar requirements for maximal activity with respect to the structural features of the biotin molecule, with one notable exception. In the case of biotin carboxylase, a free unprotonated side chain carboxyl group is required for maximal activity while the opposite is true for the carboxyltransferase-catalyzed reaction.

Acetyl Coenzyme A Carboxylase System of Escherichia coli: PURIFICATION AND PROPERTIES OF THE BIOTIN CARBOXYLASE, CARBOXYLTRANSFERASE, AND CARBOXYL CARRIER PROTEIN COMPONENTS

The three protein components (biotin carboxylase, carboxyltransferase, and the biotin-containing carboxyl carrier protein of the acetyl coenzyme A carboxylase system have been resolved and purified extensively or to homogeneity from cell-free extracts of Escherichia coli B. Carboxylation of acetyl-CoA to form malonyl-CoA requires the presence of all three components. Biotin carboxylase, which catalyzes the first half-reaction, CCP (carboxyl carrier protein)-biotin + HCO3- + (Me2+)/⇌ CCP-biotin-CO2- + ADP + Pi, (I) has been purified to a homogeneous state and has been crystallized; earlier work in this laboratory showed the enzyme to be composed of two 50,000-dalton subunits. The carboxylation of free d-biotin which can substitute for carboxyl carrier protein-biotin as model substrate is markedly activated by certain organic solvents; an optimal rate enhancement of 10-fold is obtained with 15 % (v/v) ethanol. Activation by ethanol affects Vmax and is not accompanied by changes in Km values or the state of aggregation of the enzyme. Moreover, none of the carboxyl carrier protein-dependent reactions catalyzed by biotin carboxylase, e.g. acetyl-CoA carboxylation, ATP-[14C]ADP exchange, and ATP-32Pi exchange, are activated by organic solvents. Carboxyltransferase, the catalyst for the second half-reaction, CCP-biotin-CO2- + acetyl-CoA ⇌ CCP-biotin + malonyl-CoA, (II) was purified to apparent homogeneity. Biotin carboxylase, although not required for the second half-reaction as measured by carboxyltransferase- and carboxyl carrier protein-dependent malonyl-CoA-[14C]acetyl-CoA exchange, activates this process indicating the existence of a ternary complex between the the three protein components. In addition to the above reaction (Reaction II), carboxyltransferase catalyzes: (a) net transcarboxylation from malonyl-CoA to free d-biotin derivatives in the absence of biotin carboxylase and carboxyl carrier protein, and (b) a slow biotin-independent decarboxylation of malonyl-CoA. The carboxyltransferase component has a molecular weight of 130,000 and is composed of nonidentical polypeptide chains of 30,000 and 35,000 daltons. Carboxyl carrier protein has been purified extensively by a combination of conventional methods and affinity chromatography with Sepharose-avidin (monomer). Polyacrylamide gel electrophoresis of purified carboxyl carrier protein revealed two major proteins both of which contain biotin and exhibit carboxyl carrier protein activity, i.e. carboxyl carrier protein- and carboxyltransferase-dependent malonyl-CoA-[14C]acetyl-CoA exchange. The two catalytic components of the E. coli acetyl-CoA carboxylase system, biotin carboxylase and carboxyltransferase, are devoid of free or covalently bound biotin yet have the ability to carry out their respective model half-reactions utilizing free d-biotin derivatives in place of carboxyl carrier protein. Thus, in addition to possessing binding sites for their respective substrates, each catalytic component must contain a specific binding site for the biotinyl moiety of carboxyl carrier protein. It is evident that during the over-all sequence (Reactions I + II) the caboxylated biotinyl prosthetic group must undergo translocation from the carboxylation site on biotin carboxylase to the transfer site on carboxyl transferase while remaining attached to carboxyl carrier protein through its 14 A side chain.

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 μ m at a fixed NADPH concentration of 100μ m 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.

An investigation into the role of malonyl-coenzyme A in isoprenoid biosynthesis.

1. [(14)C]Malonyl-CoA was incorporated into isoprenoids by cell-free yeast preparations, by preparations from pigeon and rat liver, and by Hevea brasiliensis latex. 2. In agreement with previous reports the incorporation of acetyl-CoA into isoprenoids was not inhibited by avidin and was not stimulated by HCO(3) (-). In a cell-free yeast preparation addition of HCO(3) (-) stimulated the formation of fatty acids from acetyl-CoA and decreased the incorporation into unsaponifiable lipids. 3. The labelling patterns of beta-hydroxy-beta-methylglutaryl-CoA formed from [2-(14)C]- and [1,3-(14)C]-malonyl-CoA in rat and pigeon liver preparations were those that would be expected if malonyl-CoA underwent decarboxylation to acetyl-CoA before incorporation. 4. The labelling pattern of ergosterol formed by cell-free yeast preparations from [2-(14)C]malonyl-CoA was also consistent with decarboxylation of malonyl-CoA before incorporation. 5. The incorporation of [2-(14)C]malonyl-CoA into mevalonate by rat liver preparations was related to the malonyl-CoA decarboxylase activity present in the preparation.