RESUMEN
In this study, we probed the inhibition of pig heart citrate synthase (E.C. 4.1.3.7) by synthesising seven analogues either designed to mimic the proposed enolate intermediate in this enzyme reaction or developed from historical inhibitors. The most potent inhibitor was fluorovinyl thioether 9 (Ki =4.3â µm), in which a fluorine replaces the oxygen atom of the enolate. A comparison of the potency of 9 with that of its non-fluorinated vinyl thioether analogue 10 (Ki =68.3â µm) revealed a clear "fluorine effect" favouring 9 by an order of magnitude. The dethia analogues of 9 and 10 proved to be poor inhibitors. A methyl sulfoxide analogue was a moderate inhibitor (Ki =11.1â µm), thus suggesting hydrogen bonding interactions in the enolate site. Finally, E and Z propenoate thioether isomers were explored as conformationally constrained carboxylates, but these were not inhibitors. All compounds were prepared by the synthesis of the appropriate pantetheinyl diol and then assembly of the coenzymeâ A structure according to a three-enzyme biotransformation protocol. A quantum mechanical study, modelling both inhibitors 9 and 10 into the active site indicated short CFâ â â H contacts of ≈2.0â Å, consistent with fluorine making two stabilising hydrogen bonds, and mimicking an enolate rather than an enol intermediate. Computation also indicated that binding of 9 to citrate synthase increases the basicity of a key aspartic acid carboxylate, which becomes protonated.
Asunto(s)
Acetilcoenzima A/análogos & derivados , Citrato (si)-Sintasa/antagonistas & inhibidores , Inhibidores Enzimáticos/química , Acetilcoenzima A/síntesis química , Animales , Dominio Catalítico , Citrato (si)-Sintasa/química , Inhibidores Enzimáticos/síntesis química , Enlace de Hidrógeno , Modelos Químicos , Teoría Cuántica , PorcinosRESUMEN
The antimetabolite pentyl pantothenamide has broad spectrum antibiotic activity but exhibits enhanced activity against Escherichia coli. The PanDZ complex has been proposed to regulate the pantothenate biosynthetic pathway in E. coli by limiting the supply of ß-alanine in response to coenzyme A concentration. We show that formation of such a complex between activated aspartate decarboxylase (PanD) and PanZ leads to sequestration of the pyruvoyl cofactor as a ketone hydrate and demonstrate that both PanZ overexpression-linked ß-alanine auxotrophy and pentyl pantothenamide toxicity are due to formation of this complex. This both demonstrates that the PanDZ complex regulates pantothenate biosynthesis in a cellular context and validates the complex as a target for antibiotic development.
Asunto(s)
Acetilcoenzima A/metabolismo , Carboxiliasas/metabolismo , Escherichia coli K12/metabolismo , Proteínas de Escherichia coli/metabolismo , Glutamato Descarboxilasa/metabolismo , Modelos Moleculares , Acetilcoenzima A/análogos & derivados , Acetilcoenzima A/química , Sustitución de Aminoácidos , Antibacterianos/farmacología , Antimetabolitos/farmacología , Sitios de Unión , Calorimetría , Carboxiliasas/química , Carboxiliasas/genética , Coenzima A/síntesis química , Coenzima A/química , Coenzima A/metabolismo , Cristalografía por Rayos X , Activación Enzimática/efectos de los fármacos , Escherichia coli K12/efectos de los fármacos , Escherichia coli K12/crecimiento & desarrollo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Eliminación de Gen , Glutamato Descarboxilasa/antagonistas & inhibidores , Glutamato Descarboxilasa/química , Glutamato Descarboxilasa/genética , Cinética , Mutación , Ácido Pantoténico/análogos & derivados , Ácido Pantoténico/farmacología , Conformación Proteica , Multimerización de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , VolumetríaRESUMEN
The p300 and CBP transcriptional coactivator paralogs (p300/CBP) regulate a variety of different cellular pathways, in part, by acetylating histones and more than 70 non-histone protein substrates. Mutation, chromosomal translocation, or other aberrant activities of p300/CBP are linked to many different diseases, including cancer. Because of its pleiotropic biological roles and connection to disease, it is important to understand the mechanism of acetyl transfer by p300/CBP, in part so that inhibitors can be more rationally developed. Toward this goal, a structure of p300 bound to a Lys-CoA bisubstrate HAT inhibitor has been previously elucidated, and the enzyme's catalytic mechanism has been investigated. Nonetheless, many questions underlying p300/CBP structure and mechanism remain. Here, we report a structural characterization of different reaction states in the p300 activity cycle. We present the structures of p300 in complex with an acetyl-CoA substrate, a CoA product, and an acetonyl-CoA inhibitor. A comparison of these structures with the previously reported p300/Lys-CoA complex demonstrates that the conformation of the enzyme active site depends on the interaction of the enzyme with the cofactor, and is not apparently influenced by protein substrate lysine binding. The p300/CoA crystals also contain two poly(ethylene glycol) moieties bound proximal to the cofactor binding site, implicating the path of protein substrate association. The structure of the p300/acetonyl-CoA complex explains the inhibitory and tight binding properties of the acetonyl-CoA toward p300. Together, these studies provide new insights into the molecular basis of acetylation by p300 and have implications for the rational development of new small molecule p300 inhibitors.
Asunto(s)
Acetilcoenzima A/análogos & derivados , Acetilcoenzima A/química , Factores de Transcripción p300-CBP/química , Dominio Catalítico , Coenzima A/química , Humanos , Modelos Moleculares , Unión Proteica , Conformación Proteica , Factores de Transcripción p300-CBP/antagonistas & inhibidoresRESUMEN
The N1-acetylation of spermidine and spermine by spermidine/spermine acetyltransferase (SSAT) is a crucial step in the regulation of the cellular polyamine levels in eukaryotic cells. Altered polyamine levels are associated with a variety of cancers as well as other diseases, and key enzymes in the polyamine pathway, including SSAT, are being explored as potential therapeutic drug targets. We have expressed and purified human SSAT in Escherichia coli and characterized its kinetic and chemical mechanism. Initial velocity and inhibition studies support a random sequential mechanism for the enzyme. The bisubstrate analogue, N1-spermine-acetyl-coenzyme A, exhibited linear, competitive inhibition against both substrates with a true Ki of 6 nM. The pH-activity profile was bell-shaped, depending on the ionization state of two groups exhibiting apparent pKa values of 7.27 and 8.87. The three-dimensional crystal structure of SSAT with bound bisubstrate inhibitor was determined at 2.3 A resolution. The structure of the SSAT-spermine-acetyl-coenzyme A complex suggested that Tyr140 acts as general acid and Glu92, through one or more water molecules, acts as the general base during catalysis. On the basis of kinetic properties, pH dependence, and structural information, we propose an acid/base-assisted reaction catalyzed by SSAT, involving a ternary complex.
Asunto(s)
Acetiltransferasas/química , Acetiltransferasas/metabolismo , Acetilcoenzima A/análogos & derivados , Acetilcoenzima A/metabolismo , Acetiltransferasas/genética , Secuencia de Bases , Cristalografía por Rayos X , ADN Complementario/genética , Humanos , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Cinética , Modelos Moleculares , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espermina/análogos & derivados , Espermina/metabolismo , Especificidad por SustratoRESUMEN
Analogues of coenzyme A (CoA) and of CoA thioesters have been prepared in which the amide bond nearest the thiol group has been modified. An analogue of acetyl-CoA in which this amide bond is replaced with an ester linkage was a good substrate for the enzymes carnitine acetyltransferase, chloramphenicol acetyltransferase, and citrate synthase, with K(m) values 2- to 8-fold higher than those of acetyl-CoA and V(max) values from 14 to >80% those of the natural substrate. An analogue in which an extra methylene group was inserted between the amide bond and the thiol group showed less than 4-fold diminished binding to the three enzymes but exhibited less than 1% activity relative to acetyl-CoA with carnitine acetyltransferase and no measurable activity with the other two enzymes. Analogues of several CoA thioesters in which the amide bond was replaced with a hemithioacetal linkage exhibited no measurable activity with the appropriate enzymes. The results indicate that some aspects of the amide bond and proper distance between this amide and the thiol/thioester moiety are critical for activity of CoA ester-utilizing enzymes.
Asunto(s)
Acetilcoenzima A/análogos & derivados , Amidas/química , Coenzima A/química , Acetilcoenzima A/síntesis química , Coenzima A/metabolismo , Ésteres/síntesis química , Estructura Molecular , Compuestos de Sulfhidrilo/químicaRESUMEN
The potential contribution of thiolimidate formation to the increased kinetic acidity of the alpha-proton of acetyl-CoA in the carbon-carbon bond forming reaction catalyzed by 3-ketoacyl-CoA thiolase (thiolase I) from porcine heart was assessed by chemical modification and isotope exchange experiments. Thiolase is only partially inactivated after the chemical modification of lysine residues by reductive methylation, pyridoxal phosphate, or o-phthaldehyde (specific for vicinal lysine and cysteine). The thiolase-catalyzed formation of acetyl-CoA from acetoacetyl-CoA and CoASH in 18OH2 is not accompanied by the appearance of 18O in the acetyl-CoA product. These experiments effectively rule out participation of thiolimidate formation in the thiolase reaction. Other mechanisms must be employed to facilitate the abstraction of the alpha-proton of acetyl-CoA by thiolase I.
Asunto(s)
Acetilcoenzima A/análogos & derivados , Acetil-CoA C-Acetiltransferasa/metabolismo , Acetiltransferasas/metabolismo , Acilcoenzima A , Miocardio/enzimología , Acetilcoenzima A/metabolismo , Acetil-CoA C-Acetiltransferasa/antagonistas & inhibidores , Animales , Sitios de Unión , Hidrólisis , Lisina , Espectrometría de Fluorescencia , Relación Estructura-Actividad , PorcinosRESUMEN
Two kinds of 3-ketoacyl-CoA thiolases were found in the peroxisomes of Candida tropicalis cells grown on n-alkanes (C10-C13). One was a typical acetoacetyl-CoA thiolase specific only to acetoacetyl-CoA, while another was 3-ketoacyl-CoA thiolase showing high activities on the longer chain substrates. A high level of the latter thiolase activity in alkane-grown cells was similar to that of other enzymes constituting the fatty acid beta-oxidation system in yeast peroxisomes. These facts suggest that the complete degradation of fatty acids to acetyl-CoA is carried out in yeast peroxisomes by the cooperative contribution of acetoacetyl-CoA thiolase and 3-ketoacyl-CoA thiolase.
Asunto(s)
Acetil-CoA C-Acetiltransferasa/metabolismo , Acetil-CoA C-Aciltransferasa/metabolismo , Acetiltransferasas/metabolismo , Acilcoenzima A , Aciltransferasas/metabolismo , Alcanos/farmacología , Candida/enzimología , Microcuerpos/enzimología , Acetilcoenzima A/análogos & derivados , Acetilcoenzima A/metabolismo , Candida/ultraestructura , Ácidos Grasos/metabolismo , Isoenzimas/metabolismo , Oxidación-ReducciónRESUMEN
Kinetics of inactivation of general acyl-CoA dehydrogenase from pig kidney by methylenecyclopropaneacetyl-CoA have been analyzed using the theoretical treatment and exact steady-state kinetic solutions reported by Tatsunami (Tatsunami, S., Yago, N., and Hosoe, M. (1981) Biochim. Biophys. Acta 662, 226-235). Thus practical application of these analytical solutions for an important class of enzyme:substrate reactions has been demonstrated for the first time.
Asunto(s)
Acetilcoenzima A/análogos & derivados , Acil-CoA Deshidrogenasas/antagonistas & inhibidores , Acetilcoenzima A/farmacología , Animales , Sitios de Unión , Riñón/enzimología , Cinética , Modelos Teóricos , Unión Proteica , PorcinosRESUMEN
The biosynthetic thiolase, from Zoogloea ramigera, involved in generation of acetoacetyl-CoA for poly-beta-hydroxybutyrate synthesis, has been prepared pure in quantity for initial structural characterization of this homotetrameric enzyme. Edman degradation provided the sequence of the NH2 terminal 25 residues and an active site cysteine-containing nonapeptide labeled on stoichiometric inactivation by iodoacetamide. Both sequences were used to align the encoding DNA sequence of the cloned gene as described in an accompanying paper. Synthetic analogs of acetoacetyl-S-CoA, modified in the CoA moiety, were prepared and tested, and acetoacetyl-S-pantetheine 11-pivalate 1 was shown to have a kcat/Km of 6.4 X 10(6) M-1 s-1, comparable to the kcat/Km of 2 X 10(7) M-1 s-1 for acetoacetyl-S-CoA. The pantetheine pivalate group facilitates nonaqueous synthetic manipulations and may be generally useful as a CoA replacement. We have also prepared the carba analog of 1, with CH2 replacing S, to yield a beta-diketone analog 10 of acetoacetyl-S-CoA and the corresponding methyl ketone analog 9 of acetyl-S-CoA. These analogs have been used to prove the ability of Z. ramigera thiolase to catalyze proton abstraction from the C-2 methyl group of the acetyl portion of substrate in a transition state separate from C-C bond formation. NMR studies in D2O show exchange only when condensation is possible. Further studies with [2-3H]acetyl-CoA show there is neither pre-equilibrium washout nor detectable kH/kT expressed in turnover and provide no evidence for a discrete acetyl-CoA C-2 carbanion or a nonconcerted reaction.
Asunto(s)
Acetil-CoA C-Acetiltransferasa/metabolismo , Acetiltransferasas/metabolismo , Acilcoenzima A , Protones , Zoogloea/enzimología , Acetilcoenzima A/análogos & derivados , Acetilcoenzima A/metabolismo , Acetil-CoA C-Acetiltransferasa/antagonistas & inhibidores , Secuencia de Aminoácidos , Fenómenos Químicos , Química , Dietil Pirocarbonato/farmacología , Yodoacetamida/farmacología , Punto Isoeléctrico , Cinética , Sustancias Macromoleculares , Peso Molecular , Especificidad por SustratoRESUMEN
The thiolase involved in biosynthesis of poly-beta-hydroxybutyrate in Zoogloea ramigera generates an acetyl-enzyme species during catalysis. Up to 0.86 [14C] acetyl eq/subunit of this homotetrameric enzyme is accumulated by acid precipitation in the presence of [14C]acetyl-CoA. Gel filtration of the same solutions produced only 7% acetyl-enzyme suggesting hydrolytic lability of the acetyl-enzyme during the 10-min isolation at 4 degrees C. In an effort to identify active site residues which may function as basic groups to deprotonate at C-2 of acetyl-CoA to generate the required nucleophilic equivalent in carbon-carbon bond formation, we have prepared and tested haloacetyl-thioesters, oxoesters, and amides in the panthetheine pivalate series (Davis, J. T., Moore, R. N., Imperiali, B., Pratt, A. J., Kobayashi, K., Masamune, S., Sinskey, A. J., and Walsh, C. T. (1987) J. Biol. Chem. 262, 82-89). The [14C]bromoacetyl-oxoester alkylatively inactivates thiolase irreversibly with stoichiometric incorporation of four labels/tetramer. Determination of amino acid composition of the radiolabeled tryptic peptide indicated trapping of Cys-89 (Peoples, O. P., Masamune, S., Walsh, C. T., and Sinskey, A. J. (1987) J. Biol. Chem. 262, 97-102), the same residue modified by iodoacetamide. When the bromoacetyl-thioester was used, inactivation was pH-dependent. The data are consistent with the competition of two processes, acylation, and alkylation. Direct (rather than secondary) alkylation of thiolase by the inactivator accounts for the significant 14C incorporation into thiolase with the thioester labeled with [14C] in the pantetheine pivalate moiety. It appears likely that the haloacetyl analogs described herein should be generally useful for affinity labeling other enzymes using acetyl-CoA as a substrate.
Asunto(s)
Acetilcoenzima A/análogos & derivados , Acetil-CoA C-Acetiltransferasa/antagonistas & inhibidores , Acetiltransferasas/antagonistas & inhibidores , Halógenos , Zoogloea/enzimología , Acetilcoenzima A/metabolismo , Acetil-CoA C-Acetiltransferasa/metabolismo , Acilación , Marcadores de Afinidad , Alquilación , Amidas , Aminoácidos/análisis , Sitios de Unión , Ésteres , Cinética , Panteteína/análogos & derivados , Panteteína/farmacología , Ácidos Pentanoicos/farmacología , Fragmentos de Péptidos/análisisRESUMEN
The enzyme phenylacetyl-CoA: 6-Aminopenicillanic acid acyltransferase of Penicillium chrysogenum was evaluated by direct bioassay against Micrococcus luteus ATCC 9341. The enzyme required dithiothreitol, was inactivated by 0.2 mM Hg2+ (100%), Zn2+ (80%), Cu2+ (60%), 1 mM N-ethylmaleimide (80%), and showed maximal catalytic activity at pH 8.4 and 20 degrees C. The V50 values for phenylacetyl-CoA and 6-aminopenicillanic acid were 0.55 mM and 1 microM, respectively. When octanoyl-CoA was employed as substrate similar results were obtained. In both cases the product generated showed strong antibacterial activity which was quickly lost when incubation was carried out with beta-lactamase. Reactions performed in the presence of Escherichia coli penicillin acylase did not generated active products when phenylacetyl-CoA was the substrate; they did with octanoyl-CoA. Time-course experiments revealed that the highest enzyme levels are found in 36 hours mycelium and remained almost constant from 48 to 96 hours; thereafter the level of the enzyme slowly decreased.
Asunto(s)
Aciltransferasas/metabolismo , Proteínas de Unión a las Penicilinas , Penicillium chrysogenum/enzimología , Penicillium/enzimología , Acetilcoenzima A/análogos & derivados , Acetilcoenzima A/metabolismo , Bioensayo , Ditiotreitol/farmacología , Cinética , Pruebas de Sensibilidad Microbiana , Micrococcus/efectos de los fármacos , Ácido Penicilánico/metabolismo , Penicilina G/biosíntesisRESUMEN
beta-Ketoacyl-CoA thiolase (acyl-CoA:acetyl-CoA C-acyltransferase, EC 2.3.1.16) is known to possess sulfhydryl groups of cysteines at the active site that are essential for its catalytic activity. Other groups at the active site that participate in the catalytic process were identified by using anhydride reagents which covalently modify the protein by specifically reacting with any amino groups potentially present at the active site. Since these reagents may also react with thiol groups, the enzyme's amino groups were modified after masking the cysteine thiols present by an alkylalkane thiosulfonate-type reagent, methyl methanethiol-sulfonate (MMTS), that selectively formed a disulfide bridge, thus generating an inactive thiolmethylated enzyme. When this procedure was followed, the enzyme could be undoubtedly modified at its amino by the anhydride reagent, leading to a doubly modified protein. The thiomethyl group could then be removed by reduction with dithiothreitol, yielding an enzyme modified solely on the amino residues. The amino group could be unblocked in turn by exposure to acidic pH. The different anhydrides inactivated thiolase, but only acetoacetyl coenzyme A (AcAcCoA) provided any protection against inactivation. When thiolmethylcitraconyl thiolase was reduced with dithiothreitol the enzyme remained inactive, but when the doubly modified enzyme was exposed to pH 5 then the reduction led to formation of an active enzyme. These results are interpreted as demonstrating a role for an amino group at the enzyme active site. A catalytic mechanism is proposed for the enzyme which involves the amino group.
Asunto(s)
Acetil-CoA C-Acetiltransferasa/metabolismo , Acetiltransferasas/metabolismo , Acilcoenzima A , Acetilcoenzima A/análogos & derivados , Acetil-CoA C-Acetiltransferasa/antagonistas & inhibidores , Aminas/metabolismo , Anhídridos/farmacología , Animales , Sitios de Unión , Catálisis , Cisteína/metabolismo , Citosol/enzimología , Concentración de Iones de Hidrógeno , Hígado/enzimología , Metilmetanosulfonato/análogos & derivados , Metilmetanosulfonato/farmacología , Ratas , Compuestos de Sulfhidrilo/metabolismo , PorcinosRESUMEN
Two patients have been studied in whom the activity of the short chain-length-specific mitochondrial 3-ketothiolase was found to be deficient. Use of a range of 3-ketoacyl-CoA substrates showed that the other 3-ketothiolase isoenzymes were normal in each case. Both patients had episodic ketosis and metabolic acidosis. One patient had substantial evidence of damage to the central nervous system and two siblings who had died of the disease. The organic aciduria was characterized by the excretion of 2-methyl-3-hydroxybutyric acid and tiglyglycine. In one patient the organic aciduria was very subtle and was masked during the presence of ketosis, but it was clarified by an isoleucine load after recovery from ketosis.
Asunto(s)
Acetil-CoA C-Aciltransferasa/deficiencia , Aciltransferasas/deficiencia , Ácido 3-Hidroxibutírico , Acetilcoenzima A/análogos & derivados , Acetilcoenzima A/metabolismo , Acetil-CoA C-Aciltransferasa/metabolismo , Acilcoenzima A/orina , Niño , Femenino , Fibroblastos/enzimología , Glicina/análogos & derivados , Glicina/orina , Humanos , Hidroxibutiratos/orina , Lactante , Isoleucina , Cetosis/metabolismo , Masculino , Valeratos/metabolismoRESUMEN
The application of radiolabeled fatty acids to measurements of fatty acid oxidation is discussed and a method for measuring the rate of beta-oxidation and of acetyl-CoA oxidation to CO2 is described. In hepatocytes from starved or fed rats, ethanol inhibited total beta-oxidation in the presence of 1.3 mM palmitate by 22% and 25%, respectively. If changes in the specific radioactivity of acetyl-CoA were not corrected for, the effect of ethanol would have been overestimated by 15% and underestimated by 15% in hepatocytes from fed and starved rats, respectively. In perfused liver from fed rats, inhibition by ethanol of total beta-oxidation in the presence of 1 mM palmitate was 35%. The rate of beta-oxidation in the absence of ethanol was underestimated by 65% if proper corrections were not applied. Inhibition of the tricarboxylic acid cycle by ethanol was 57% and 72% in hepatocytes from starved and fed rats, respectively. Pyrazole titration experiments demonstrated a correlation between changes in the mitochondrial NADH/NAD+ ratio and both inhibition of the tricarboxylic acid cycle and inhibition of the beta-oxidation pathway. The concentration of acetoacetyl-CoA is suggested as an additional regulatory factor of the beta-oxidation pathway. The ethanol-induced accumulation of triacylglycerol as a consequence of the inhibition of the beta-oxidation pathway is estimated to represent a 10% increase in the cellular triacylglycerol pool/hr/g of wet weight. Hence its chemical determination requires experiments of several hours duration. Primary cultures of hepatocytes have been shown to be a useful experimental system for studies of the ethanol-induced triacylglycerol accumulation.
Asunto(s)
Acilcoenzima A , Etanol/farmacología , Ácidos Grasos/metabolismo , Hígado/metabolismo , Acetilcoenzima A/análogos & derivados , Acetilcoenzima A/análisis , Animales , Células Cultivadas , Ciclo del Ácido Cítrico/efectos de los fármacos , Técnicas In Vitro , Mitocondrias Hepáticas/análisis , NAD/análisis , Oxidación-Reducción , Ratas , Triglicéridos/biosíntesisRESUMEN
The mechanism of acyl enzyme formation from acyl-CoA derivatives was studied for chicken liver fatty acid synthase in 0.1 M potassium phosphate (pH 7.0) and 1 mM EDTA at 23 degrees C. Three mechanistically important acyl-binding sites exist: a cysteine, 4'-phosphopantetheine, and a hydroxyl (serine). The cysteine was specifically labeled with iodoacetamide, and chemical modification of this labeled enzyme with chloroacetyl-CoA resulted in additional covalent labeling of 4'-phosphopantetheine. Reaction of the enzyme with acetyl-CoA results in 47% oxyester formation, whereas with malonyl-CoA and butyryl-CoA, 57 and 80% are oxyesters, respectively, as judged by treatment of the denatured enzyme with hydroxylamine. Limited proteolysis with trypsin followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that the reactive hydroxyl and cysteine are on the same peptide. Butyryl-CoA is a relatively poor primer for steady state fatty acid synthesis, probably because transfer from the hydroxyl-binding site to 4'-phosphopantetheine is inefficient. Quenched flow studies indicate that the rate constants for transfer of acetyl from enzyme-bound acetyl-CoA to native, iodoacetamide-labeled, and iodoacetamide-chloroacetyl-labeled enzyme are 43, 110, and 150 s-1. These results can be interpreted in terms of a random acylation of the hydroxyl, 4'-phosphopantetheine, and cysteine by enzyme-bound acetyl-CoA with rate constants of 150 s-1, less than 110 s-1, and less than 43 s-1, respectively. Alternatively the latter two rate constants could be characteristic of intramolecular transfer between enzyme acylation sites. Structural constraints apparently prevent all three acylation sites from being occupied simultaneously. The rate of deacetylation of the acetylated enzyme by enzyme-bound CoA also is most rapid for the iodoacetamide-chloroacetyl-labeled enzyme.
Asunto(s)
Ácido Graso Sintasas/metabolismo , Hígado/enzimología , Acetilcoenzima A/análogos & derivados , Acetilcoenzima A/metabolismo , Acilación , Animales , Sitios de Unión , Pollos , Ácido Graso Sintasas/antagonistas & inhibidores , Concentración de Iones de Hidrógeno , Yodoacetamida/farmacología , CinéticaRESUMEN
The overt form of carnitine palmitoyltransferase (CPT1) in rat liver and heart mitochondria was inhibited by DL-2-bromopalmitoyl-CoA and bromoacetyl-CoA. S-Methanesulphonyl-CoA inhibited liver CPT1. The inhibitory potency of DL-2-bromopalmitoyl-CoA was 17 times greater with liver than with heart CPT1. Inhibition of CPT1 by DL-2-bromopalmitoyl-CoA was unaffected by 5,5'-dithiobis-(2-nitrobenzoic acid) or (in liver) by starvation. In experiments in which DL-2-bromopalmitoyl-CoA displaced [14C]malonyl-CoA bound to liver mitochondria, the KD (competing) was 25 times the IC50 for inhibition of CPT1 providing evidence that the malonyl-CoA-binding site is unlikely to be the same as the acyl-CoA substrate site. Bromoacetyl-CoA inhibition of CPT1 was more potent in heart than in liver mitochondria and was diminished by 5,5'-dithiobis-(2-nitrobenzoic acid) or (in liver) by starvation. Bromoacetyl-CoA displaced bound [14C]malonyl-CoA from heart and liver mitochondria. In heart mitochondria this displacement was competitive with malonyl-CoA and was considerably facilitated by L-carnitine. In liver mitochondria this synergism between carnitine and bromoacetyl-CoA was not observed. It is suggested that bromoacetyl-CoA interacts with the malonyl-CoA-binding site of CPT1. L-Carnitine also facilitated the displacement by DL-2-bromopalmitoyl-CoA of [14C]malonyl-CoA from heart, but not from liver, mitochondria. DL-2-Bromopalmitoyl-CoA and bromoacetyl-CoA also inhibited overt carnitine octanoyl-transferase in liver and heart mitochondria. These findings are discussed in relation to inter-tissue differences in (a) the response of CPT1 activity to various inhibitors and (b) the relationship between high-affinity malonyl-CoA-binding sites and those sites for binding of L-carnitine and acyl-CoA substrates.
Asunto(s)
Acetilcoenzima A/análogos & derivados , Acilcoenzima A/análogos & derivados , Acilcoenzima A/metabolismo , Aciltransferasas/antagonistas & inhibidores , Carnitina O-Palmitoiltransferasa/antagonistas & inhibidores , Malonil Coenzima A/metabolismo , Mitocondrias/metabolismo , Palmitoil Coenzima A/análogos & derivados , Acetilcoenzima A/farmacología , Animales , Sitios de Unión , Carnitina Aciltransferasas/metabolismo , Coenzima A/análogos & derivados , Coenzima A/farmacología , Cinética , Mitocondrias Cardíacas/efectos de los fármacos , Mitocondrias Cardíacas/enzimología , Mitocondrias Hepáticas/efectos de los fármacos , Mitocondrias Hepáticas/enzimología , Palmitoil Coenzima A/farmacología , RatasRESUMEN
Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (EC 4.1.3.5) was purified to homogeneity from ox liver and obtained essentially free from acetoacetyl-CoA thiolase activity. The purification procedure included substrate elution from cellulose phosphate and chromatofocusing. The relative molecular mas was about 100 000 and S20,w0 was 6.36S. The enzyme appears to be a dimer of identical subunits (Mr 47 900). The Km for acetoacetyl-CoA is extremely low (less than 0.5 microM), and acetoacetyl-CoA (Acac-CoA) gives marked substrate inhibition (KiAcac-CoA = 3.5 microM) that is competitive with respect to acetyl-CoA. Both CoA and DL-3-hydroxy-3-methylglutaryl-CoA give mixed product inhibition with respect to acetyl-CoA, which is compatible with a Ping Pong mechanism in which both products can form kinetically significant complexes with two forms of the enzyme. The two forms are most likely to be free enzyme and an acetyl-enzyme intermediate.
Asunto(s)
Acilcoenzima A , Hidroximetilglutaril-CoA Sintasa/aislamiento & purificación , Mitocondrias Hepáticas/enzimología , Oxo-Ácido-Liasas/aislamiento & purificación , Acetilcoenzima A/análogos & derivados , Acetilcoenzima A/metabolismo , Aminoácidos/análisis , Animales , Bovinos , Cromatografía en Gel , Electroforesis en Gel de Poliacrilamida , Hidroximetilglutaril-CoA Sintasa/metabolismo , Líquido Intracelular/enzimología , Cinética , Peso Molecular , UltracentrifugaciónRESUMEN
Ox liver mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (EC 4.1.3.5) reacts with acetyl-CoA to form a complex in which the acetyl group is covalently bound to the enzyme. This acetyl group can be removed by addition of acetoacetyl-CoA or CoA. The extent of acetylation and release of CoA were found to be highly temperature-dependent. At temperatures above 20 degrees C, a maximum value of 0.85 mol of acetyl group bound/mol of enzyme dimer was observed. Below this temperature the extent of rapid acetylation was significantly lowered. Binding stoichiometries close to 1 mol/mol of enzyme dimer were also observed when the 3-hydroxy-3-methylglutaryl-CoA synthase activity was titrated with methyl methanethiosulphonate or bromoacetyl-CoA. This is taken as evidence for a 'half-of-the-sites' reaction mechanism for the formation of 3-hydroxy-3-methylglutaryl-CoA by 3-hydroxy-3-methylglutaryl-CoA synthase. The Keq. for the acetylation was about 10. Isolated acetyl-enzyme is stable for many hours at 0 degrees C and pH 7, but is hydrolysed at 30 degrees C with a half-life of 7 min. This hydrolysis is stimulated by acetyl-CoA and slightly by succinyl-CoA, but not by desulpho-CoA. The site of acetylation has been identified as the thiol group of a reactive cysteine residue by affinity-labelling with the substrate analogue bromo[1-14C]acetyl-CoA.