Phosphopantetheine Prosthetic Group Research Articles

Expression of holo and apo forms of spinach acyl carrier protein-I in leaves of transgenic tobacco plants.

Acyl carrier protein (ACP) is a chloroplast-localized cofactor of fatty acid synthesis, desaturation, and acyl transfer. We have transformed tobacco with a chimeric gene consisting of the tobacco ribulose-1,5-bisphosphate carboxylase promoter and transit peptide and the sequence encoding the mature spinach ACP-I. Spinach ACP-I was expressed in the transformed plants at levels twofold to threefold higher than the endogenous tobacco ACPs as determined by protein immunoblots and assays of ACP in leaf extracts. In addition to these elevated levels of the holo form, there were high levels of apoACP-I, a form lacking the 4'-phosphopantetheine prosthetic group and not previously detected in vivo. The mature forms of both apoACP-I and holoACP-I were located in the chloroplasts, indicating that the transit peptide was cleaved and that attachment of the prosthetic group was not required for uptake into the plastid. There were also significant levels of spinach acyl-ACP-I, demonstrating that spinach ACP-I participated in tobacco fatty acid metabolism. Lipid analyses of the transformed plants indicated that the increased ACP levels caused no significant alterations in leaf lipid biosynthesis.

The function of acyl carrier protein in the synthesis of membrane-derived oligosaccharides does not require its phosphopantetheine prosthetic group.

An enzyme system catalyzing the synthesis of the beta 1,2-linked glucan backbone of the membrane-derived oligosaccharides of Escherichia coli from UDP-glucose has an essential requirement for the E. coli acyl carrier protein (ACP). This finding was surprising, because all other characterized functions of ACP involve acyl thioester residues linked to the phosphopantetheine moiety covalently bound to ACP. We now report that the activity of ACP in the synthesis of membrane-derived oligosaccharides is not altered by treatment with the sulfhydryl reagent N-ethylmaleimide nor by complete removal of the phosphopantetheine residue by treatment with a specific phosphodiesterase. The function of ACP in the synthesis of membrane-derived oligosaccharides is thus clearly different from that involved in lipid biosynthesis. We have nevertheless found that the same molecular species of ACP that undergo enzymic acylation with long-chain fatty acid residues also function in the synthesis of membrane-derived oligosaccharides.

Synthesis, cloning, and expression in Escherichia coli of a spinach acyl carrier protein-I gene

A synthetic gene of 268 bp encoding the 82 amino acid spinach acyl carrier protein (ACP)-I was constructed based on the known amino acid sequence. Two gene fragments, one encoding the amino-terminal portion and the other the car☐y-terminal portion of the protein, were assembled from synthetic oligonucleotides and inserted into the phage M13mp19. These partial gene constructions were joined and inserted into the plasmid pTZ19R. DNA sequencing confirmed the accuracy of the constructions. The synthetic gene was then subcloned into the Escherichia coli expression vector pKK233-2, under the control of the trc promoter. Western blot analysis and radioimmunoassay indicated that E. coli cells carrying this plasmid produced up to 6 mg/liter of a protein which was immunologically cross-reactive and similar in electrophoretic mobility to authentic spinach acyl carrier protein. The bacterial cells were able to attach the phosphopantetheine prosthetic group to the synthetic plant gene product allowing it to be acylated in vitro by acyl-ACP synthetase.

Purification and characterization of Rhodobacter sphaeroides acyl carrier protein.

Acyl carrier protein (ACP) has been purified from the facultative phototrophic bacterium Rhodobacter sphaeroides. The ACP preparation was greater than 95% homogeneous as determined by native and disodium dodecyl sulfate (Na2DodSO4)-polyacrylamide gel electrophoreses and N-terminal amino acid analysis. Amino acid compositional analysis revealed that the protein contains approximately 75 amino acids, has a calculated minimum molecular weight of 8700, and lacks the amino acids tyrosine and tryptophan. The presence of the characteristic 4'-phosphopantetheine prosthetic group was indicated by the occurrence of equimolar quantities of beta-alanine and taurine in amino acid hydrolysates and was confirmed by independent chemical analysis. The protein displayed a pI of 3.8 and had a calculated partial specific volume of 0.732 mL/g. The primary structure of the protein has been determined for the first 46 amino acid residues from the N terminus of the molecule, and the region of the molecule encompassing the amino acids from residues 31 to 44 was found to have 100% homology with the identical residues in Escherichia coli ACP. In contrast to E. coli ACP, R. sphaeroides ACP migrated according to its molecular weight during Na2DodSO4 gel electrophoresis, was resistant to pH-induced denaturation, and comigrated with the cis-vaccenoyl-ACP derivative during native gel electrophoresis. It is proposed that the basis for these properties is the enhanced hydrophobic character of the protein.

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.

Altered molecular form of acyl carrier protein associated with beta-ketoacyl-acyl carrier protein synthase II (fabF) mutants.

Acyl carrier protein (ACP) is a required cofactor for fatty acid synthesis in Escherichia coli. Mutants lacking beta-ketoacyl-ACP synthase II activity (fabF1 or fabF3) possessed a different molecular species of ACP (F-ACP) that was separated from the normal form of the protein by conformationally sensitive gel electrophoresis. Synthase I mutants contained the normal protein. Complementation of fabF1 mutants with an F' factor harboring the wild-type synthase II allele resulted in the appearance of normal ACP, whereas complementation with an F' possessing the fabF2 allele (a mutation that produces a synthase II enzyme with altered catalytic activity) resulted in the production of both forms of ACP. The structural difference between F-ACP and ACP persisted after the removal of the 4'-phosphopantetheine prosthetic group, and both forms of the protein had identical properties in an in vitro fatty acid synthase assay. Both ACP and F-ACP were purified to homogeneity, and their primary amino acid sequences were determined. The two ACP species were identical but differed from the sequence reported for E. coli E-15 ACP in that an Asn instead of an Asp was at position 24 and an Ile instead of a Val was at position 43. Therefore, F-ACP appears to be a modification of ACP that is detected when beta-ketoacyl-ACP synthase II activity is impaired.

Turnover of the 4'-phosphopantetheine prosthetic group of acyl carrier protein.

Acyl carrier protein is an essential cofactor in fatty acid biosynthesis, and in contrast to the stability of the protein moiety during growth, its 4'-phosphopantetheine prosthetic group is metabolically active. The biosynthetic incorporation of deuterium into nonexchangeable positions of acyl carrier protein was found to enhance the sensitivity of the protein to pH-induced hydrodynamic expansion. This constitutional isotope effect was exploited to separate deuterated from normal acyl carrier protein by conformationally sensitive gel electrophoresis, thus providing the analytical framework for separating pre-existing (deuterated) from newly synthesized acyl carrier protein in pulse-chase experiments. The rate of acyl carrier protein prosthetic group turnover was found to depend on the intracellular concentration of coenzyme A. At low coenzyme A levels, prosthetic group turnover was four times faster than the rate of new acyl carrier protein biosynthesis but at the higher coenzyme A concentrations characteristic of logarithmic growth, turnover was an order of magnitude slower, amounting to approximately 25% of the acyl carrier protein pool per generation. These observations suggest that the acyl carrier protein prosthetic group turnover cycle may be related to coenzyme A metabolism rather than to lipid biosynthesis.

Ratio of active to inactive forms of acyl carrier protein in Escherichia coli.

Acyl carrier protein (ACP) functions as a cofactor in fatty acid biosynthesis due to the covalent linkage of an acyl moiety to its 4'-phosphopantetheine prosthetic group. This prosthetic group undergoes turnover in vivo and since the apoprotein is functionally inactive, the interconversion between ACP and apo-ACP has been considered as a possible regulatory point in lipid biosynthesis. To investigate this possibility, the ratio of ACP to apo-ACP was measured in Escherichia coli. An apo-ACP standard was synthesized using [ACP] phosphodiesterase (EC 3.1.4.14) and could be clearly separated from ACP by conformationally sensitive gel electrophoresis, thus providing a reliable assay for the presence of these two species. Antibodies specific for ACP were purified from rabbit serum on an ACP-Sepharose column and subsequently used to synthesize an immunoaffinity column. Chromatography of leucine-labeled cell extracts on this support resulted in the specific binding of ACP, but apo-ACP was not detected in either logarithmically growing or stationary phase cells, although both ACP species bound to the purified anti-ACP IgG. Apo-ACP was not detected as an intermediate in ACP biosynthesis, suggesting that apo-ACP is rapidly converted to ACP following translation. CoA is the biosynthetic precursor to the ACP prosthetic group, but apo-ACP did not accumulate when the intracellular CoA concentration was severely depressed in strain SJ16 (panD), a beta-alanine auxotroph. Strain MP4 (acpS) is conditionally defective in [ACP]synthase (EC 2.7.8.7) and apo-ACP was the predominant form of ACP synthesized in this strain under nonpermissive conditions. Even under conditions that permitted growth, apo-ACP comprised 70% of the total ACP pool in strain MP4. Strain MP4 possessed a phospholipid to protein ratio within the normal range, suggesting that the ratio of ACP to apo-ACP can be significantly altered without affecting total lipid content. Thus, it appears that the prosthetic group turnover cycle maintains all of the ACP in an active form in vivo and a regulatory role for the ACP/apo-ACP ratio seems doubtful.

The architecture of the animal fatty acid synthetase. III. Isolation and characterization of beta-ketoacyl reductase.

Sequential treatment of the chicken liver fatty acid synthetase by trypsin and subtilisin cleaved the Mr 267,000 subunit to 6-8 polypeptides, ranging in molecular weights from 15,000 to 94,000. Fractionation of the digest by ammonium sulfate and chromatography on a Procion Red HE3B affinity column permitted the isolation of a polypeptide (Mr = 94,000) containing the beta-ketoacyl reductase activity but no other partial activities normally associated with the synthetase. The specific activity of the beta-ketoacyl reductase increased 2 to 3 times in this fraction, an increase that is within the expected range based on relative molecular weight. The kinetic parameters of this fraction towards NADPH and N-acetyl-S-acetoacetyl cysteamine were essentially the same as the beta-ketoacyl reductase component of the intact synthetase. However, the purified fragment did not catalyze the reduction of acetoacetyl-S-CoA derivative, a substrate that is readily reduced by the intact synthetase. Fluorescence measurements with etheno-NADP+ indicate the binding of about 1 mol of NADP+/94,000 daltons, a value which is in agreement with the results obtained from fluorescence measurements with NADPH and the binding of a radiolabeled photoaffinity analog of NADP+. Phenylglyoxal inhibits the beta-ketoacyl reductase activity of either the intact synthetase or the isolated fragment, suggesting the involvement of an essential arginine at or near the active site. Another fragment (Mr 36,000) containing beta-ketoacyl reductase activity was isolated from the synthetase after kallikrein/subtilisin double digestion. Previous mapping studies had shown that this fragment lies adjacent to the COOH-terminal thioesterase domain and overlaps the tryptic Mr 94,000 peptide by approximately 21 daltons. This fragment, but not the Mr 94,000 fragment, was found to contain the phosphopantetheine prosthetic group, indicating that the acyl carrier protein moiety is located in the 15,000-dalton segment that separates the beta-ketoacyl reductase from the thioesterase domain.

The architecture of the animal fatty acid synthetase complex. IV. Mapping of active centers and model for the mechanism of action.

The fatty acid synthetase of animal tissue consists of two subunits, each containing seven catalytic centers and an acyl carrier site. Proteolytic cleavage patterns indicate that the subunit is arranged into three major domains, I, II, and III. Domain I contains the NH2-terminal end of the polypeptide and the catalytic sites of beta-ketoacyl synthetase (condensing enzyme) and the acetyl-and malonyl-transacylases. This domain, therefore, functions as a site for acetyl and malonyl substrate entry into the process of fatty acid synthesis and acts in part as the site of carbon-carbon condensation, resulting in chain elongation. Domain II is the medial domain and contains the beta-ketoacyl and enoyl reductases, probably the dehydratase, and the 4'-phosphopantetheine prosthetic group of the acyl carrier protein site. Domain II, therefore, is designated as the reduction domain where the keto carbon is reduced to methylene carbon by sequential processes of reduction, dehydration, and reduction again. Throughout these processes, the acyl group is attached to the pantetheine-SH of the acyl carrier protein. The latter site is distal to the cysteine-SH of the beta-ketoacyl synthetase, constitutes the 15000-dalton polypeptide at the COOH-terminal end of Domain II, and connects to Domain III. When the growing chain reaches C16 carbon length, the fatty acyl group is released by the thioesterase activity, which is contained in Domain III. A functional model is proposed based on the aforementioned results and the recent evidence that the synthetase subunits are arranged in a head-to-tail fashion, such that the pantetheine-SH of the acyl carrier protein of one subunit and the cysteine-SH of the beta-ketoacyl synthetase of the second subunit are juxtaposed. In this model, a palmitate synthesizing site contains Domain I of one subunit and Domains II and III of the second subunit. Therefore, even though each subunit contains all of the partial activities of the reaction sequence, the actual palmitate synthesizing unit consists of one-half of a subunit interacting with the complementary half of the other subunit.

Molecular properties of acyl carrier protein derivatives.

Acyl carrier protein (ACPSH) functions as the acyl carrier in fatty acid biosynthesis. The acyl moieties are bound to the sole sulfhydryl of the protein located on the 4'-phosphopantetheine prosthetic group. Disulfide-linked dimers of ACPSH were formed by the reaction of ACPSH with acyl-ACP or the mixed disulfide of ACPSH and thionitrobenzoate. The formation of ACP dimers was established by electrophoresis, gel filtration, and sedimentation equilibrium. ACP purified from stationary phase Escherichia coli B cells was found to exist primarily as a mixed disulfide with glutathione. This species was identified by gel electrophoresis amino acid analysis and 31P NMR spectroscopy. A non-denaturing gel electrophoresis system was developed that allows the comparison of the effects of various protein and sulfhydryl modifications on the stability of the ACP protein moiety to pH-induced denaturation. In general, attachment of hydrophilic ligands to the sulfhydryl of ACPSH resulted in less stable protein structures whereas the presence of a hydrophobic thioester resulted in stabilization of the protein conformation. The less stable ACP structures were found to have 31P NMR chemical shifts displaced downfield from ACPSH and the more stable acyl-ACP derivatives were found to have chemical shifts displaced upfield from ACPSH.

Isolation, purification, and properties of mammalian and avian liver and yeast fatty acid synthetase acyl carrier proteins

Acyl carrier proteins were isolated from rat, human, pigeon, and chicken liver and yeast fatty acid synthetase complexes. These proteins were separated from the other proteins of subunit I of each complex by ultrafiltration after dialysis of subunit I for 3 h against low ionic strength buffer [Qureshi et al. (1974) Biochem. Biophys. Res. Commun. 60, 158–165]. Subunit I of each fatty acid synthetase was previously separated from subunit II by affinity chromatography on Sepharose ϵ-aminocaproyl pantetheine and subsequent sucrose density gradient centrifugation. The separated acyl carrier proteins were then subjected to gel filtration on a Sephadex G-50 column. The proteins obtained from each fatty acid synthetase were homogeneous with respect to size and charge on gel filtration, paper and disc gel electrophoresis, and chromatography on diethylaminoethyl-cellulose. The physical properties and the ability to accept acetyl and malonyl groups from acetyl- and malonyl-CoA in the presence of transacylase were similar to those of Escherichia coli acyl carrier protein. These proteins ranged in molecular weight from 7500 to 10,000. Each of the acyl carrier proteins showed the presence of β-alanine and each yielded acetyl- and malonyl-A 1 and A 2 peptic peptides, thus indicating the presence of a 4′-phosphopantetheine prosthetic group in each. They differed somewhat from each other in amino acid composition, but each had a high number of negatively charged (aspartate and glutamate) amino acid residues.

Occurrence of apoAcyl carrier protein and an S-Alkylation-resistant form of holoAcyl carrier protein in Escherichia coli

The 4′-phosphopantetheine prosthetic group of holoacyl carrier protein (holoACP) in Escherichia coli turns over independently of the apoprotein, due to the activities of holoACP hydrolase and holoACP synthetase. There is no measurable pool of apoACP in pantothenate-supplemented cells of a pantothenate-requiring mutant, but extended incubation on deficient medium, with exhaustion of cellular coenzyme A (CoA), leads to slow accumulation of the apoprotein. It is concluded that, although the activities of the synthetase and hydrolase are about equal in crude extracts, in the cells an excess synthetase activity maintains ACP completely as holoACP unless cells are artifically depleted of CoA, the donor of the 4′-phosphopantetheine group. About 20% of the holoACP in normal cells was designated as “holoACP esters,” being resistant to S-alkylation unless first treated with neutral hydroxylamine; this proportion increased to about 80% in pantothenate starvation. A preliminary attempt to identify acyl portions from this material was unsuccessful. The proportion of this material was not elevated in other strains under conditions which show feedback inhibition of fatty acid biosynthesis in vivo.

Adaptive synthesis of rat liver fatty acid synthetase: Evidence for [formula omitted] formation of active enzyme from inactive protein precursors and 4′-phosphopantetheine

Evidence is presented in support of the hypothesis that an important step in the adaptive synthesis of fatty acid synthetase is the conversion of inactive enzyme precursors to active enzyme via the incorporation of the 4′-phosphopantetheine prosthetic group. Fatty acid synthetase activity was generated in vitro when CoA or E. coli acyl carrier protein was incubated with enzymatically inactive extracts from livers of rats fed a fat-free diet for 0–5 hr following starvation, and a factor present in liver extracts from rats refed for more than 6 hr. When ( 14C)-CoA, labelled in the pantetheine moiety, was used in the above system, radioactivity was incorporated into a protein bound form, from which it could be released by mild alkaline hydrolysis.

Studies on the adaptive synthesis of rat liver fatty acid synthetase

The adaptive synthesis of fatty acid synthetase in the livers of rats fed a fat-free diet following 48 hr of fasting has been studied using immunochemical methods. The development of fatty acid synthetase activity during adaptive synthesis occurs about 3 hr following feeding, whereas the synthesis of material precipitable by anti-fatty acid synthetase serum, as judged by the incorporation of 3H-labeled amino acids into the immunoprecipitate, commenced within 1 hr. Extracts of liver of rats fed a fat-free diet for 1–3 hr following fasting contain increasing amounts of material which competes with purified fatty acid synthetase for antibody binding sites, even though they have no fatty acid synthetase activity. This suggests the presence of enzymatically inactive precursors of fatty acid synthetase in the liver extracts. The incorporation of [ 14C]pantothenate into fatty acid synthetase during adaptive synthesis follows the same pattern as the development of enzyme activity, indicating that these enzymatically inactive precursors of fatty acid synthetase may represent an apoenzyme which is converted to the enzymatically active holoenzyme by the incorporation of the 4′-phosphopantetheine prosthetic group. The subcellular site of synthesis of fatty acid synthetase was shown to be in the pool of polysomes that are not membrane bound, rather than in the rough endoplasmic reticulum.

Fatty acid synthetase of mammalian brain, liver and adipose tissue regulation by prosthetic group turnover

The role of prosthetic group turnover in the regulation of the fatty acid synthetase of mammalian brain, adipose tissue and liver has been studied. The fatty acid synthetase was labeled in the 4'-phosphopantetheine prosthetic group of the acyl carrier protein by administration to rats of calcium [ 3H]pantothenate. By determining the time course of incorporation of radioactivity into both CoA, the precursor for the 4'-phosphopantetheine moiety of acyl carrier protein, and the fatty acid synthetase, it was possible to estimate the rate of prosthetic group turnover. The turnover rate was found to be extremely rapid in all three tissues. In brain, the prosthetic group is replaced completely in 10–11 h and this is an order of magnitude faster than the turnover of the enzyme complex, previously shown to occur with a t 1 4 of 6.4 days. In liver and adipose tissue, the prosthetic group turnover is slightly faster than in brain. Thus, the rapid turnover of the prosthetic group, first shown in Escherichia coli, also occurs in three different mammalian tissues and may represent a general biological phenomenon. Since turnover of the protein of fatty acid synthetase of brain does not change at all in various nutritional states when turnover of liver synthetase changes dramatically, we considered the question of whether the striking turnover of the prosthetic group in these tissues also responded differently to those states. Distinct changes in the efficiency of the exchange of the 4'-phosphopantetheine between CoA and fatty acid synthetase were observed only in the liver but not in the brain. Increased exchange in liver during fat-free feeding was discovered and the earlier observation of diminished exchange during starvation was confirmed. We also evaluated by immunochemical methods the possibility that rapid regulation of fatty acid synthetase is mediated by changes in catalytic efficiency. Quantitative precipitin analyses of liver extracts from animals during the first hours after onset of starvation or upon refeeding after starvation demonstrated that changes in enzyme activity were related entirely to changes in enzyme content. Thus, the data did not support the possibility that fatty acid synthetase activity is decreased by changes in catalytic efficiency due to removal of the 4'-phosphopantetheine prosthetic group or to inhibition by metabolites.

The Stability of Acyl Carrier Protein in Escherichia coli

Abstract Two techniques have been employed to examine the turnover of the protein portion of acyl carrier protein, the conjugated protein which is the acyl carrier for fatty acid biosynthesis in Escherichia coli. The protein was shown to be metabolically stable, in contrast to its covalently bound 4'-phosphopantetheine prosthetic group which was previously found to undergo turnover at the rate of 4% per min (Powell, G. L., Elovson, J., and Vagelos, P. R. (1969) J. Biol. Chem. 244, 5616–5624).

Studies on the Mechanism of Fatty Acid Synthesis: XXV. ON THE MECHANISM OF β-KETOACYL ACYL CARRIER PROTEIN REDUCTASE FROM ESCHERICHIA COLI

Abstract Although highly purified preparations of β-ketoacyl acyl carrier protein (ACP) reductase are obtained from extracts of Escherichia coli, purification is hampered by the enzyme's instability in dilute solution. An investigation of this phenomenon revealed that concentrations of TPNH and TPN+ below 1 mm and those of multivalent anions between 0.2 m and 1 m could protect the active form of the enzyme by binding to the protein and thereby stabilizing its active form. Enzyme inactivation occurred in two stages, the first being reversible by TPNH, TPN+, and multivalent anions, and the second being irreversible by these agents. In these studies potassium phosphate acted as an inhibitor of TPNH oxidation, while potassium chloride failed to alter the reaction rate, indicating that TPNH, TPN+, and phosphate may be bound to the same site (possibly the catalytic site) of the enzyme. From a comparative kinetic study with a number of model substrates, it was concluded that the substrate, acetoacetyl-ACP was bound to β-ketoacyl-ACP reductase through its protein moiety, the pantoyl residue of the 4'-phosphopantetheine prosthetic group, and the β-ketoester group. ACP was a competitive inhibitor of the reduction of acetoacetyl-ACP, acetoacetyl pantetheine and acetoacetyl-N-acetyl-cysteamine, emphasizing the importance of the ACP-protein-enzyme interaction for substrate binding and demonstrating the inaccessibility of the catalytic site for model substrates once the ACP-protein is bound to the enzyme. Since the enzyme reduced ethyl thiolacetoacetate and ethyl acetoacetate, we concluded that the cysteaminyl residue and the thioester linkage of the acetoacetyl substrate were not absolutely necessary for the substrate-enzyme binding. However, the absence of the cysteaminyl residue in the substrate significantly decreased the Vmax values. The fact that acetoacetyl-ACP in its enolate form would apparently not bind to the enzyme suggests that the keto form of the substrate is directly reduced in this reaction.

Intermediates of fatty acid synthesis: Sites of binding to the pigeon liver fatty acid synthetase

The pigeon-liver fatty acid synthetase complex contains three acetyl- and two malonyl-binding sites. One of these sites (B 1) appears to be a hydroxyl-containing compound. Acetyl and malonyl groups (transferred from their CoA esters) are bound to this site in increased amounts when the sulfhydryl sites that bind acyl groups to the enzyme are blocked with iodoacetamide. This result suggests that B 1 is a primary or loading site for acetyl and malonyl groups. Competitive inhibition studies have shown that acetyl and malonyl groups compete for this site, thereby suggesting that the fatty acid synthetase may have a single loading site (B 1) for these groups. Similarly, acetyl and malonyl groups bind competitively to the 4′-phosphopantetheine (A 2) prosthetic group of the enzyme. A third site (B 2) binds acetyl, but not malonyl, groups to the enzyme. The acetoacetyl moiety is bound to the 4′-phosphopantetheine (A 2) site of the enzyme. The formation of the acetoacetyl group on this site has been shown to be deendent on the availability of the B 2 site for the binding of the acetyl group. A reduction of acetyl binding to this site, by selective iodoacetamide inhibition resulted in a parallel reduction in the formation of acetoacetyl-enzyme. Incubation of the enzyme with malonyl-CoA and hexanoyl-CoA yields β-ketooctanoyl-enzyme. The β-ketooctanoyl moiety is bound to the enzyme as a thioester of 4′-phosphopantetheine. Reduction of β-ketooctanoyl-enzyme in the presence of NADPH yields octanoyl-enzyme as a product. The octanoyl group is bound to the enzyme through thioester bonding at both the A 2 (4′-phosphopantetheine) and B 2 sites. The location of intermediates on specific prosthetic groups of the enzyme is utilized to deduce both the order of the intermediate reactions and the role played by each prosthetic group during fatty acid synthesis.

Synthesis of Triacetic Acid Lactone by the Pigeon Liver Fatty Acid Synthetase Complex

The synthesis of triacetic acid lactone (TAL) is effected by purified soluble pigeon liver fatty acid synthetase. This enzyme system synthesizes TAL from acetyl coenzyme A and malonyl coenzyme A in the absence of TPNH, and synthesizes palmitic acid in the presence of TPNH. The major product of the reaction in the absence of TPNH is TAL, and not free triacetic acid. Presumably triacetic acid, bound as a thioester to the 4′-phosphopantetheine prosthetic group of the fatty acid synthetase, is an intermediate in the formation of TAL. The latter compound is formed from 1 mole of acetyl-CoA and 2 moles of malonyl-CoA in the sequence, acetate-malonate-malonate (proceeding left to right from the methyl end of the molecule). The synthesis of TAL is inhibited by either TPN+ or TPNH. The very strong inhibition by both forms of the nucleotide implies that TAL is not synthesized in vivo by the avian soluble fatty acid synthetase.