RESUMEN
Computational studies were performed in an effort to understand the relative reactivity of oxoesters and thioesters in nucleophilic acyl transfer reactions. Transition state models were developed for the reactions of methyl acetate and methyl thioacetate with hydroxide, ammonia, and methylcyanoacetate carbanion. Quantum mechanical calculations based on these models reproduced experimental observations that oxoesters and thioesters have similar reactivity toward hydroxide while thioesters are about 100-fold and at least 2000-fold more reactive than oxoesters toward amine and carbanion nucleophiles, respectively. NBO analysis was performed to elucidate the role of electron delocalization in reactant and transition state stabilization. These calculations indicate similar losses of delocalization energy for the oxoester and thioester in going from the reactants to the transition state in reaction with hydroxide while the loss of delocalization energy is significantly greater for the oxoester in reactions with the other nucleophiles. Bond rotational analysis of the transition states for the reactions with hydroxide and ammonia provide support for an important role of the p(X) --> sigma(C-Nu) interaction (X = O or S of the oxoester or thioester respectively, Nu = nucleophile) in governing the reactivity of oxoesters and thioesters in nucleophilic acyl substitution.
Asunto(s)
Acetatos/química , Amoníaco/química , Hidróxidos/química , Modelos Moleculares , Nitrilos/química , TermodinámicaRESUMEN
Dephosphocoenzyme A (dephospho-CoA) kinase catalyzes the final step in coenzyme A biosynthesis, the phosphorylation of the 3'-hydroxy group of the ribose sugar moiety. Wild-type dephospho-CoA kinase from Corynebacterium ammoniagenes was purified to homogeneity and subjected to N-terminal sequence analysis. A BLAST search identified a gene from Escherichia coli previously designated yacE encoding a highly homologous protein. Amplification of the gene and overexpression yielded recombinant dephospho-CoA kinase as a 22.6-kDa monomer. Enzyme assay and nuclear magnetic resonance analyses of the product demonstrated that the recombinant enzyme is indeed dephospho-CoA kinase. The activities with adenosine, AMP, and adenosine phosphosulfate were 4 to 8% of the activity with dephospho-CoA. Homologues of the E. coli dephospho-CoA kinase were identified in a diverse range of organisms.
Asunto(s)
Proteínas Bacterianas/genética , Escherichia coli/genética , Genes Bacterianos , Genes , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Adenosina/metabolismo , Adenosina Monofosfato/metabolismo , Adenosina Fosfosulfato/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Clonación Molecular , Corynebacterium/enzimología , Escherichia coli/enzimología , Escherichia coli/metabolismo , Datos de Secuencia Molecular , Fosfotransferasas (Aceptor de Grupo Alcohol)/aislamiento & purificación , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Proteínas Recombinantes/genética , Alineación de SecuenciaRESUMEN
An analogue 2 of coenzyme A (CoA) has been prepared in which the geminal methyl groups are replaced with hydrogens. An NMR titration study was conducted and shifts in frequency of protons in the pantetheine portion of the molecule upon titration of the adenine base were observed as has been previously reported with CoA. These studies indicate that the geminal dimethyl groups are not essential for adoption of a partially folded conformation in solution. Based on 1H-1H coupling constants, the distribution of conformations about the carbon-carbon bonds in the region of the methyl deletion were estimated. The results suggest that the conformer distribution is similar to that of CoA, but with small increases in population of the anti conformers. A simple model compound containing the didemethyl pantoamide moiety was prepared and subjected to similar conformational analysis. The coupling constants and predicted conformer distribution were almost identical to that of the CoA analogue, indicating that the conformer distribution is controlled by local interactions and not influenced by interactions between distant parts of the CoA molecule. The acetyl derivative of 2 was a fairly good substrate for the acetyl-CoA utilizing enzymes carnitine acetyltransferase, chloramphenicol acetyltransferase, and citrate synthase, with 1.3- to 10-fold increased Km values and 2.5- to 11-fold decreases in Vmax. The combined results indicate that the geminal dimethyl groups of CoA have modest effects on function and minimal effects on conformation.
Asunto(s)
Coenzima A/química , Carnitina O-Acetiltransferasa/química , Carnitina O-Acetiltransferasa/metabolismo , Cloranfenicol O-Acetiltransferasa/química , Cloranfenicol O-Acetiltransferasa/metabolismo , Espectroscopía de Resonancia Magnética , Estructura Molecular , Conformación ProteicaRESUMEN
The ionization state and hydrogen bonding environment of the transition state analogue (TSA) inhibitor, carboxymethyldethia coenzyme A (CMX), bound to citrate synthase have been investigated using solid state NMR. This enzyme-inhibitor complex has been studied in connection with the postulated contribution of short hydrogen bonds to binding energies and enzyme catalysis: the X-ray crystal structure of this complex revealed an unusually short hydrogen bond between the carboxylate group of the inhibitor and an aspartic acid side chain [Usher et al. (1994) Biochemistry 33, 7753-7759]. To further investigate the nature of this short hydrogen bond, low spinning speed 13C NMR spectra of the CMX-citrate synthase complex were obtained under a variety of sample conditions. Tensor values describing the chemical shift anisotropy of the carboxyl groups of the inhibitor were obtained by simulating MAS spectra (233 +/- 4, 206 +/- 5, and 105 +/- 2 ppm vs TMS). Comparison of these values with our previously reported database and ab initio calculations of carbon shift tensor values clearly indicates that the carboxyl is deprotonated. New data from model compounds suggest that hydrogen bonds in a syn arrangement with respect to the carboxylate group have a pronounced effect upon the shift tensors for the carboxylate, while anti hydrogen bonds, regardless of their length, apparently do not perturb the shift tensors of the carboxyl group. Thus the tensor values for the enzyme-inhibitor complex could be consistent with either a very long syn hydrogen bond or an anti hydrogen bond; the latter would agree very well with previous crystallographic results. Two-dimensional 1H-13C heteronuclear correlation spectra of the enzyme-inhibitor complex were obtained. Strong cross-peaks were observed from the carboxyl carbon to proton(s) with chemical shift(s) of 22 +/- 5 ppm. Both the proton chemical shift and the intensity of the cross-peak indicate a very short hydrogen bond to the carboxyl group of the inhibitor, the C.H distance based upon the cross-peak intensity being 2.0 +/- 0.4 A. This proton resonance is assigned to Hdelta2 of Asp 375, on the basis of comparison with crystal structures and the fact that this cross-peak was absent in the heteronuclear correlation spectrum of the inhibitor-D375G mutant enzyme complex. In summary, our NMR studies support the suggestion that a very short hydrogen bond is formed between the TSA and the Asp carboxylate.
Asunto(s)
Citrato (si)-Sintasa/antagonistas & inhibidores , Citrato (si)-Sintasa/química , Inhibidores Enzimáticos/química , Acilcoenzima A/química , Sustitución de Aminoácidos/genética , Animales , Ácido Aspártico/genética , Sitios de Unión/genética , Ácidos Carboxílicos/química , Citrato (si)-Sintasa/genética , Glicina/genética , Enlace de Hidrógeno , Iones , Mutagénesis Sitio-Dirigida , Resonancia Magnética Nuclear Biomolecular , Ácido Oxaloacético/química , Protones , PorcinosRESUMEN
In 1997 and the first half of 1998, numerous publications appeared reporting studies of cofactors and their analogues in classical model systems and in enzyme-catalyzed reactions directed at understanding the enzymatic reactions of their natural cofactors. Model systems based on flavins have provided new insights into enzymatic modulation of the flavin reduction potential, and enzymatic reactions of coenzyme A analogues and derivatives have been employed in several studies of coenzyme A utilizing enzymes. Coenzyme B12 analogues have been utilized as alternate cofactors for B12-utilizing enzymes, while pyrroloquinoline quinone esters and analogues have been employed in model studies of the reactions of quinoprotein-catalyzed reactions.
Asunto(s)
Coenzimas/metabolismo , Modelos BiológicosRESUMEN
The catalytic strategies of enzymes (such as citrate synthase) whose reactions require the abstraction of the alpha-proton of a carbon acid remain elusive. Citrate synthase readily catalyzes solvent proton exchange of the methyl protons of dethiaacetyl-coenzyme A, a sulfur-less, ketone analog of acetyl-coenzyme A, in its ternary complex with oxaloacetate. Because no further reaction occurs with this analog, it provides a uniquely simple probe of the roles of active site interactions on carbon acid proton transfer catalysis. In view of the high reactivity of the analog for proton transfer to the active site base, its failure to further condense with oxaloacetate to form a sulfur-less analog of citryl-coenzyme A was unexpected, although we offer several possible explanations. We have measured the rate constants for exchange, k(exch), at saturating concentrations of the analog for six citrate synthase mutants with single changes in active site residues. Comparisons between the values of k(exch) are straightforward in two limits. If the rate of exchange of the transferred proton with solvent protons is rapid, then k(exch) equals the forward rate constant for proton transfer, and k(exch) values for different mutants compare directly the rate constants for proton transfer. If the exchange of the transferred proton with protons in the bulk solution is the slow step and the equilibrium constant for proton transfer is unfavorable (as is likely), then k(exch) equals the product of the equilibrium constant for proton transfer and the rate constant for exchange of the transferred proton with bulk solvent. If that exchange rate with bulk solution remains constant for a series of mutant enzymes, then k(exch) values compare the equilibrium constants for proton transfer. The importance of the acetyl-CoA site residues, H274 and D375, is confirmed with D375 again implicated as the active site base. The results with the series of oxaloacetate site mutants, H320X, strongly suggest that activation of the first substrate, oxaloacetate, through carbonyl bond polarization, not just oxaloacetate binding in the active site, is required for the enzyme to efficiently catalyze proton transfer from the methyl group of the second substrate.
Asunto(s)
Acetilcoenzima A/metabolismo , Citrato (si)-Sintasa/metabolismo , Acetilcoenzima A/química , Animales , Sitios de Unión , Dicroismo Circular , Citrato (si)-Sintasa/genética , Clonación Molecular , Cartilla de ADN/química , Escherichia coli/genética , Expresión Génica/genética , Cinética , Espectroscopía de Resonancia Magnética , Estructura Molecular , Mutación , Miocardio/enzimología , Oxaloacetatos/metabolismo , Protones , PorcinosRESUMEN
INTRODUCTION: The polyketide natural products are assembled by a series of decarboxylation/condensation reactions of simple carboxylic acids catalyzed by polyketide synthase (PKS) complexes. The growing chain is assembled on acyl carrier protein (ACP), an essential component of the PKS. ACP requires posttranslational modification on a conserved serine residue by covalent attachment of a 4'-phosphopantetheine (P-pant) cofactor to yield active holo-ACP. When ACPs of Streptomyces type II aromatic PKS are overproduced in E. coli, however, typically little or no active holo-ACP is produced, and the ACP remains in the inactive apo-form. RESULTS: We demonstrate that E. coli holo-ACP synthase (ACPS), a fatty acid biosynthesis enzyme, can catalyze P-pant transfer in vitro to the Streptomyces PKS ACPs required for the biosynthesis of the polyketide antibiotics granaticin, frenolicin, oxytetracycline and tetracenomycin. The catalytic efficiency of this P-pant transfer reaction correlates with the overall negative charge of the ACP substrate. Several coenzyme A analogs, modified in the P-pant portion of the molecule, are likewise able to serve as substrates in vitro for ACPS. CONCLUSIONS: E coli ACPS can serve as a useful reagent for the preparation of holo-forms of Streptomyces ACPs as well as holo-ACPs with altered phosphopantetheine moieties. Such modified ACPs should prove useful for studying the role of particular ACPs and the phosphopantetheine cofactor in the subsequent reactions of polyketide and fatty acid biosynthesis.
Asunto(s)
Proteína Transportadora de Acilo/metabolismo , Coenzima A/metabolismo , Escherichia coli/enzimología , Streptomyces/metabolismo , Transferasas (Grupos de Otros Fosfatos Sustitutos)/metabolismo , Antibacterianos/metabolismo , Clonación Molecular , Cartilla de ADN , Estructura Molecular , Complejos Multienzimáticos/metabolismo , Panteteína/análogos & derivados , Panteteína/metabolismo , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Transferasas (Grupos de Otros Fosfatos Sustitutos)/biosíntesis , Transferasas (Grupos de Otros Fosfatos Sustitutos)/aislamiento & purificaciónRESUMEN
Recent work in the synthesis of cyclic phosphonate analogues of glucose [Darrow, J.W.; Drueckhammer, D.G. (1994) J. Org. Chem. 1994, 59, 2976] has been extended to the synthesis of a corresponding phosphonamidate analogue. A phosphonate salt, phosphonate methyl ester, and phosphonamidate analogue were tested as inhibitors of two inverting alpha-glycosidases, (trehalase and glucoamylase), and two retaining glycosidases, (alpha-glucosidase and beta-glucosidase). No inhibition of any of these enzymes was observed with the phosphonate salt or methyl ester. However, the phosphonamidate gave moderate competitive inhibition of the two inverting glycosidases and the retaining alpha-glucosidase but no inhibition of beta-glucosidase. The phosphonamidate showed enhanced binding relative to a simple monosaccharide only with the inverting glycosidases. This enhanced binding is believed to be due to hydrogen bonding interactions between the phosphonamidate group and two active site carboxylate residues implicated in catalysis. The selectivity toward inverting glycosidases is consistent with differences in distance of an active site carboxylate from the anomeric carbon of the glycoside substrate for the inverting versus the retaining glycosidases.
Asunto(s)
Glucosa/análogos & derivados , Glicósido Hidrolasas/antagonistas & inhibidores , Animales , Glucano 1,4-alfa-Glucosidasa/antagonistas & inhibidores , Inhibidores de Glicósido Hidrolasas , Riñón/enzimología , Cinética , Porcinos , Trehalasa/antagonistas & inhibidores , beta-Glucosidasa/antagonistas & inhibidoresRESUMEN
An alpha-fluoro acid analog and an alpha-fluoro amide analog of acetyl-CoA have been synthesized. The ternary complexes of these inhibitors with oxaloacetate and citrate synthase have been crystallized and their structures analyzed at 1.7 A resolution. The structures are similar to those reported for the corresponding non-fluorinated analogs (Usher et al., 1994), with all forming unusually short hydrogen bonds to Asp 375. The alpha-fluoro amide analog binds with an affinity 1.5-fold lower than that of a previously described amide analog lacking the alpha-fluoro group. The alpha-fluoro acid analog binds with a 50-fold decreased affinity relative to the corresponding unfluorinated analog. The binding affinities are consistent with increased strengths of hydrogen bonds to Asp 375 with closer matching of pKa values between hydrogen bond donors and acceptors. The results do not support any direct correlation between hydrogen bond strength and hydrogen bond length in enzyme-inhibitor complexes.
Asunto(s)
Acetilcoenzima A/química , Citrato (si)-Sintasa/antagonistas & inhibidores , Inhibidores Enzimáticos/química , Compuestos de Flúor/química , Acetilcoenzima A/síntesis química , Acetilcoenzima A/metabolismo , Compuestos de Flúor/síntesis química , Compuestos de Flúor/metabolismo , Enlace de Hidrógeno , Conformación ProteicaRESUMEN
Two extremely potent inhibitors of citrate synthase, carboxyl and primary amide analogues of acetyl coenzyme A, have been synthesized. The ternary complexes of these inhibitors with oxaloacetate and citrate synthase have been crystallized and their structures analyzed at 1.70- and 1.65-A resolution, respectively. The inhibitors have dissociation constants in the nanomolar range, with the carboxyl analogue binding more tightly (Ki = 1.6 nM at pH 6.0) than the amide analogue (28 nM), despite the unfavorable requirement for proton uptake by the former. The carboxyl group forms a shorter hydrogen bond with the catalytic Asp 375 (distance < 2.4 A) than does the amide group (distance approximately 2.5 A). Particularly with the carboxylate inhibitor, the very short hydrogen bond distances measured suggest a low barrier or short strong hydrogen bond. However, the binding constants differ by only a factor of 20 at pH 6.0, corresponding to an increase in binding energy for the carboxyl analogue on the enzyme of about 2 kcal/mol more than the amide analogue, much less than has been proposed for short strong hydrogen bonds based on gas phase measurements [> 20 kcal/mol (Gerlt & Gassman, 1993a,b)]. The inhibitor complexes support proposals that Asp 375 and His 274 work in concert to form an enolized form of acetyl-coenzyme A as the first step in the reaction.
Asunto(s)
Acetilcoenzima A/química , Citrato (si)-Sintasa/antagonistas & inhibidores , Animales , Sitios de Unión , Pollos , Citrato (si)-Sintasa/química , Citrato (si)-Sintasa/ultraestructura , Cristalografía por Rayos X , Enlace de Hidrógeno , Técnicas In Vitro , Ligandos , Modelos Moleculares , Miocardio/enzimología , Estructura Terciaria de ProteínaRESUMEN
We have discovered that the final two steps in coenzyme A biosynthesis in Brevibacterium ammoniagenes are catalyzed by distinct enzymes, readily separated by DEAE sepharose anion exchange chromatography. This is in contrast to mammalian tissues in which these two reactions are catalyzed by a single bifunctional enzyme (Worrall, D.M., and Tubbs, P.K. (1983) Biochem. J. 215, 153-157) and Bakers yeast in which these two activities have been identified as part of a multifunctional complex (Bucovaz, E.T., Rhoades, J.L., and Tarnowski, S.J. (1980) Fed. Proc. 39 (6), 142). The pantetheine phosphate adenylyltransferase has been purified to homogeneity and found to exist as a trimeric protein of molecular mass approximately 108 kDa. Of other nucleoside triphosphates tested as substrates, only 2'-deoxy-ATP showed measurable activity, being 27% that of ATP.