Cyclohexanedione herbicides are inhibitors of rat heart acetyl-CoA carboxylase

Acetyl CoA carboxylase (ACC) catalyzes the carboxylation of acetyl CoA to form malonyl CoA. In skeletal muscle and heart, malonyl CoA functions to regulate lipid oxidation by inhibition of carnitine palmitoyltransferase-1, an enzyme which controls the entry of long chain fatty acids into mitochondria. We have found that several members of the cyclohexanedione class of herbicides are competitive inhibitors of rat heart ACC. These compounds constitute valuable reagents for drug development and the study of ACCbeta, a validated anti-obesity target.     

Interaction of acetyl-CoA fragments with rat liver acetyl-CoA carboxylase

The interaction of acetyl-CoA fragments with rat liver acetyl-CoA carboxylase has been studied. Dephosphorylated acetyl-CoA did not actually differ from acetyl-CoA in its substrate properties. Non-nucleotide analogues of the substrate, S-acetylpantatheine and it's 4'-phosphate, also possess substrate properties (Vmax = 1.5% and 15% of the maximal rate value of acetyl-CoA carboxylation, respectively). The nucleotide fragment in the acetyl-CoA molecule produces a marked effect on the thermodynamics of the substrate-enzyme interaction, and is apparently involved in activation and appropriate orientation of the acetyl group in the active site. The better substrate properties of S-acetylpantetheine 4'-phosphate and the inhibitory properties of pantetheine 4'-phosphate, compared to the unphosphorylated analogues, evidence an important role of the 5'-beta-phosphate of 3'-phosphorylated ADP residue in acetyl-CoA binding to the enzyme.     

Heat activation of rat liver acetyl-CoA carboxylase in vitro.

Acetyl-CoA carboxylase in rat liver homogenates was activated in vitro in a time- and temperature-dependent manner. The activity of acetyl-CoA carboxylase in rat liver preparations was determined in a 1-min assay to preclude the possibility of citrate activation of the enzyme during the assay period. Activation of the enzyme occurred more rapidly in liver preparations continuously maintained at ambient or greater temperatures than in homogenates of liver which had been chilled. High speed supernatant (105,000 X g, 60 min) did not heat-activate, and reconstitution of the heat-activatable 27,000 X g, 20-min, fraction by recombining the high speed pellet with the high speed supernatant only partially restored the heat activatability. Elution of the 105,000 X g supernatant from Sephadex G-25 resulted in an enzyme preparation which was heat-activatable. Addition of boiled 105,000 X g supernatant to the Sephadex G-25-treated enzyme again prevented heat activation. Dilution of the enzyme 5-fold did not prevent heat activation.     

Phosphorylation of proteolytically-nicked rat hepatic acetyl-CoA carboxylase

A partially-purified preparation of acetyl-CoA carboxylase was not inactivated by ATP and Mg2+ although it was phosphorylated. SDS gel electrophoresis of the phosphorylated enzyme showed phosphopeptides migrating at 140 and 40 K along with the 250 K native subunit. Phosphorylation by the catalytic subunit of cAMP-dependent protein kinase further phosphorylated an additional 120 K phosphopeptide. Neither cAMP-independent phosphorylation nor the cAMP-dependent phosphorylation of the enzyme resulted in a significant decrease in activity.     

The polymerization of acetyl-CoA carboxylase

Citrate, an allosteric activator of acetyl-CoA carboxylase, induces polymerization of an inactive protomeric form of the enzyme into an active filamentous form composed of 10-20 protomers. The light-scattering properties of the carboxylase were used to study the kinetics of its polymerization and depolymerization. From stopped flow kinetic studies, we have established that polymerization is a second order process, with a second order rate constant of 597,000 M-1 s-1. There appear to be two steps which limit polymerization of the inactive carboxylase protomer: 1) a rapid citrate-induced conformational change which is independent of enzyme concentration and leads to an active protomeric form of the enzyme (Beaty, N. B., and Lane, M. D. (1983) J. Biol. Chem. 258, 13043-13050, preceding paper) and 2) the dimerization of the active protomer, which constitutes the first step of polymerization and is enzyme concentration-dependent. Dimerization is the rate-limiting step of acetyl-CoA carboxylase polymerization. Depolymerization of fully polymerized acetyl-CoA carboxylase is caused by malonyl-CoA, ATP X Mg, and Mg2+. Both malonyl-CoA and ATP X Mg (and HCO-3) compete with citrate in the maintenance of a given state of the protomer-polymer equilibrium apparently by carboxylating the enzyme to form enzyme-biotin-CO-2 which destablizes the polymeric form. Free citrate is the species responsible for polymerizing the enzyme and Mg2+ causes depolymerization of the enzyme by lowering the concentration of free citrate.     

Enzymatically inactive forms of acetyl-CoA carboxylase in rat liver mitochondria.

Biotinyl proteins were labelled by incubation of SDS-denatured preparations of subcellular fractions of rat liver with [14C]methylavidin before polyacrylamide-gel electrophoresis. Fluorographic analysis showed that mitochondria contained two forms of acetyl-CoA carboxylase [acetyl-CoA:carbon dioxide ligase (ADP-forming) EC 6.4.1.2], both of which were precipitated by antibody to the enzyme. When both forms were considered, almost three-quarters of the total liver acetyl-CoA carboxylase was found in the mitochondrial fraction of liver from fed rats while only 3.5% was associated with the microsomal fraction. The remainder was present in cytosol, either as the intact active enzyme or as a degradation product. The actual specific activity of the cytosolic enzyme was approx. 2 units/mg of acetyl-CoA carboxylase protein while that of the mitochondrial enzyme was about 20-fold lower, indicating that mitochondrial acetyl-CoA carboxylase was relatively inactive. Fractionation of mitochondria with digitonin showed that acetyl-CoA carboxylase was associated with the outer mitochondrial membrane. The available evidence suggests that mitochondrial acetyl-CoA carboxylase represents a reservoir of enzyme which can be released and activated under lipogenic conditions.     

Phosphoinositide inhibition of acetyl-CoA carboxylase from rat liver

When purified acetyl-CoA carboxylase was incubated with various phospholipids, the effects on carboxylase activity were quite diverse. Phosphatidic acid, phosphatidylcholine, and phosphatidylinositol were slightly stimulatory, whereas carboxylase was inhibited by polyphosphoinositides in a time- and concentration-dependent manner. Phosphatidylinositol 4,5-bisphosphate (TPI) was the most effective inhibitor; carboxylase activity was inhibited 50% after incubation with 1.5 μm TPI for 30 min. Incubation of carboxylase with citrate reduced the susceptibility to inhibition by TPI. The inhibition was reversed by removal of TPI from the inhibited enzyme. Incubation of TPI with divalent metal cations removed its ability to inhibit carboxylase. Sedimentation studies showed that TPI treatment shifts carboxylase to a less-polymerized form. The K_m for ATP, 24 μm, was not affected by the inhibitor. However, the apparent K_m for acetyl-CoA was decreased from 44 to 11 μm following incubation with TPI. The possibility that polyphosphoinositides may play a role in acetyl-CoA carboxylase regulation is discussed.     

Erythromycin production in Saccharopolyspora erythraea does not require a functional propionyl-CoA carboxylase

Using an oligonucleotide corresponding to the consensus sequence for the biotin-binding motif, two unlinked genetic loci, bpl1 and bpl2 , were cloned from the erythromycin producer Saccharopolyspora erythraea and the nucleotide sequences of a c . 4 kb segment from each determined. The two loci share a virtually identical segment of 1746 nucleotides, coinciding with most of the genes designated bcpA1 and bcpA2 present in bpl1 and bpl2 , respectively. The deduced sequences of these genes are highly similar to that of the α-chain of mammalian propionyl-CoA carboxylase. Upstream of bcpA2 lies pccB , the gene encoding the β-chain of this enzyme. Mutant strains carrying frameshift mutations in bcpA1 and pccB were constructed, but we failed to isolate insertional mutants in bcpA2 . Propionyl-CoA carboxylase activity was undetectable in the pccB mutant, but was unaffected in the bcpA1 -defective strain. These results indicate that pccB encodes the β-chain of propionyl-CoA carboxylases, and suggest that the α-chain of this enzyme, which is likely to be encoded by bcpA2 , is shared with some other essential biotin-dependent enzyme. The pccB mutation had no impact on erythromycin production in complex medium.     

Characterization of acetyl-CoA/propionyl-CoA carboxylase in Metallosphaera sedula. Carboxylating enzyme in the 3-hydroxypropionate cycle for autotrophic carbon fixation.

Autotrophic Archaea of the family Sulfolobaceae (Crenarchaeota) use a modified 3-hydroxypropionate cycle for carbon dioxide assimilation. In this cycle the ATP-dependent carboxylations of acetyl-CoA and propionyl-CoA to malonyl-CoA and methylmalonyl-CoA, respectively, represent the key CO2 fixation reactions. These reactions were studied in the thermophilic and acidophilic Metallosphaera sedula and are shown to be catalyzed by one single large enzyme, which acts equally well on acetyl-CoA and propionyl-CoA. The carboxylase was purified and characterized and the genes were cloned and sequenced. In contrast to the carboxylase of most other organisms, acetyl-CoA/propionyl-CoA carboxylase from M. sedula is active at 75 degrees C and is isolated as a stabile functional protein complex of 560 +/- 50 kDa. The enzyme consists of two large subunits of 57 kDa each representing biotin carboxylase (alpha) and carboxytransferase (gamma), respectively, and a small 18.6 kDa biotin carrier protein (beta). These subunits probably form an (alpha beta gamma)4 holoenzyme. It has a catalytic number of 28 s-1 at 65 degrees C and at the optimal pH of 7.5. The apparent Km values were 0.06 mm for acetyl-CoA, 0.07 mm for propionyl-CoA, 0.04 mm for ATP and 0.3 mm for bicarbonate. Acetyl-CoA/propionyl-CoA carboxylase is considered the main CO2 fixation enzyme of autotrophic members of Sulfolobaceae and the sequenced genomes of these Archaea contain the respective genes. Due to its stability the archaeal carboxylase may prove an ideal subject for further structural studies.     

Effect of epinephrine on acetyl-CoA carboxylase in rat epididymal fat tissue.

If acetyl-CoA carboxylase in epididymal fat tissue is subject to control by convalent modification as in the case of the liver enzyme, catalytically different forms of carboxylase should exist, independent of polymerization. By treating epididymal fat tissue in culture with epinephrine, we have demonstrated catalytically less active forms of acetyl-CoA carboxylase. The catalytically less active forms of the enzyme reacted to antibody with the same efficiency as the active form of carboxylase. However, the less active enzyme formed by epinephrine treatment of tissues has a sedimentation constant of 30 to 35 S, whereas that of the enzyme from control tissue is 45 S. Incubation of the less active forms of the carboxylase with 10 mM citrate and up to 10 mg/ml of bovine serum albumin activated the enzyme without any change in the sedimentation constant. Therefore, the less active forms of the carboxylase formed as a result of epinephrine treatment are not due to the depolymerization of polymeric forms (45 S) to the protomeric forms (17 to 20 S), but to the formation of intermediate species of carboxylase which cannot form polymeric enzyme (45 S) in the presence of high concentrations of citrate.     

The gene encoding the biotin carboxylase subunit of Escherichia coli acetyl-CoA carboxylase.

We report the molecular cloning and DNA sequence of the gene encoding the biotin carboxylase subunit of Escherichia coli acetyl-CoA carboxylase. The biotin carboxylase gene encodes a protein of 449 residues that is strikingly similar to amino-terminal segments of two biotin-dependent carboxylase proteins, yeast pyruvate carboxylase and the alpha-subunit of rat propionyl-CoA carboxylase. The deduced biotin carboxylase sequence contains a consensus ATP binding site and a cysteine-containing sequence preserved in all sequenced bicarbonate-dependent biotin carboxylases that may play a key catalytic role. The gene encoding the biotin carboxyl carrier protein (BCCP) subunit of acetyl-CoA carboxylase is located upstream of the biotin carboxylase gene and the two genes are cotranscribed. As previously reported by others, the BCCP sequence encoded a protein of 16,688 molecular mass. However, this value is much smaller than that (22,500 daltons) obtained by analysis of the protein. Amino-terminal amino acid sequencing of the purified BCCP protein confirmed the deduced amino acid sequence indicating that BCCP is a protein of atypical physical properties. Northern and primer extension analyses demonstrate that BCCP and biotin carboxylase are transcribed as a single mRNA species that contains an unusually long untranslated leader preceding the BCCP gene. We have also determined the mutational alteration in a previously isolated acetyl-CoA carboxylase (fabE) mutant and show the lesion maps within the BCCP gene and results in a BCCP species defective in acceptance of biotin. Translational fusions of the carboxyl-terminal 110 or 84 (but not 76) amino acids of BCCP to beta-galactosidase resulted in biotinated beta-galactosidase molecules and production of one such fusion was shown to result in derepression of the biotin biosynthetic operon.     

Reevaluation of properties of acetyl-CoA carboxylase from rat liver

Rat liver acetyl-CoA carboxylase can be rapidly isolated by a new procedure which uses avidin-Sepharose affinity chromatography. The isolated enzyme has Mr = 260,000; none or very little of the proteolytic products of the carboxylase which are formed in conventional purification procedures are found in our preparations. It is apparent that the previously reported subunit of the carboxylase, with Mr = 230,000, is itself the product of proteolysis. The properties of the enzyme produced by our new method are quite different from those of the conventionally prepared enzyme. Our enzyme contains 6 mol of alkali-labile phosphate/mol of subunit, rather than 2 mol; the Km for acetyl-CoA is about 8-fold higher and the specific activity is only about one-fifth of that previously reported. The large amount of phosphate does not appear to cause the low specific activity of the new enzyme preparation, because alkaline phosphatase treatment reduces the number of phosphates/subunit from 6 to 3 mol but does not change the specific activity.     

Hepatic zonation of acetyl-CoA carboxylase activity.

The activities of several hepatic enzymes are preferentially zonated to the periportal or perivenous cells of the liver acinus. Employing dual-digitonin-pulse perfusion of rat liver in the study of acetyl-CoA carboxylase (ACC), we have identified a heretofore unrecognized feature of hepatic zonation, namely an intrahepatic gradient in enzyme specific activity. ACC activity shows a relative periportal localization in normally feeding rats, even when corrected for ACC protein mass. In contrast with results previously reported by us [Evans, Quistorff & Witters (1989) Biochem. J. 259, 821-829], the total mass of both hepatic ACC isoenzymes was not found to differ between the two hepatic zones in the present study. In perfusion eluates from fed animals, periportal ACC displays enhanced citrate reactivity and two kinetic components of acetyl-CoA reactivity; the largest periportal/perivenous gradient (5-fold) is accounted for by a species with a lower Km for acetyl-CoA. The zonal gradient in ACC maximal velocity, measured in eluates from fed rats, does not persist after ACC purification, although the isolated periportal enzyme, like dephosphorylated ACC, has a lower activation constant for citrate. Total ACC protein phosphatase activity is higher in periportal eluates, but no differences in the activities of either a 5'-AMP-activated ACC kinase or the cyclic-AMP-dependent protein kinase are noted between the hepatic zones. The induction of total hepatic ACC mass and specific activity, on fasting/refeeding with a high-carbohydrate diet, abolishes the periportal/perivenous activity gradient, largely owing to a selective activation of perivenous enzyme. Nutritional induction is also accompanied by a marked alteration in ACC acetyl-CoA kinetics and abolition of the gradient in total ACC phosphatase. These studies indicate that hepatic enzyme zonation, which is often attributed to differential expression of enzyme protein, may result from zonal variations in enzyme specific activity, owing to differences in allosteric regulation and/or covalent modification.