Asunto(s)
Péptido Natriurético Encefálico/sangre , Resistencia Física/fisiología , Carrera/fisiología , Troponina I/sangre , Troponina T/sangre , Adulto , Creatina Quinasa/sangre , Forma MB de la Creatina-Quinasa , Cardiopatías/sangre , Humanos , Isoenzimas/sangre , Persona de Mediana Edad , Mioglobina/sangre , Tomografía Computarizada de Emisión de Fotón Único , Disfunción Ventricular Izquierda/diagnósticoRESUMEN
Inactivation of HMG-CoA synthase by a carboxyl-directed reagent, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), in a concentration-dependent and substrate-protectable manner suggested that the active site contains reactive acidic amino acids. This observation prompted functional evaluation of 11 invariant acidic amino acids by site-directed mutagenesis. Characterization of the isolated synthase variants' ability to catalyze overall and partial reactions identified three mutant synthases (D99A, D159A, and D203A) that exhibit significant diminution of k(cat) for the overall reaction (10(2)-, 10(3)-, and 10(4)-fold decreases, respectively). D99A, D159A, and D203A form the acetyl-S-enzyme intermediate very slowly (0.0025, 0.0026, 0.0015 U/mg, respectively, measured at pH 7. 0 and 22 degrees C) as compared to the wild-type synthase (1.59 U/mg), where intermediate formation approaches rate-limiting status. Differences in substrate saturation do not account for impaired activities or rates of intermediate formation. The structural integrity of the purified mutants' active sites is demonstrated by their abilities to bind a spin-labeled acyl-CoA analogue (R.CoA) with affinities and stoichiometries comparable to values measured for wild-type synthase. The impact of three distinct amino acids on reaction intermediate formation supports a mechanism of acetyl-S-enzyme formation that probably requires formation and directed collapse of a tetrahedral adduct. (18)O-induced shift of the (13)C NMR signal of (13)C acetyl-S-enzyme demonstrates that an analogous tetrahedral species is produced upon solvent exchange with the acetyl-S-enzyme. Partial discrimination between the functions of D99, D159, and D203 becomes possible based on the observation that D159A and D203A synthases exhibit retarded kinetics of solvent (18)O exchange while D99A fails to support (18)O exchange.
Asunto(s)
Acetilcoenzima A/química , Sustitución de Aminoácidos , Aminoácidos/química , Hidroximetilglutaril-CoA Sintasa/química , Acetilación , Alanina/genética , Sustitución de Aminoácidos/genética , Aminoácidos/genética , Animales , Ácido Aspártico/genética , Catálisis , Inhibidores Enzimáticos/química , Etildimetilaminopropil Carbodiimida/química , Ácido Glutámico/genética , Humanos , Concentración de Iones de Hidrógeno , Hidrólisis , Hidroximetilglutaril-CoA Sintasa/antagonistas & inhibidores , Hidroximetilglutaril-CoA Sintasa/genética , Hidroximetilglutaril-CoA Sintasa/aislamiento & purificación , Mutagénesis Sitio-Dirigida , Isótopos de Oxígeno , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Solventes , AguaRESUMEN
Replacement of 3-hydroxy-3-methylglutaryl-CoA synthase's glutamate 95 with alanine diminishes catalytic activity by over 5 orders of magnitude. The structural integrity of E95A enzyme is suggested by the observation that this protein contains a full complement of acyl-CoA binding sites, as indicated by binding studies using a spin-labeled acyl-CoA. Active site integrity is also demonstrated by (13)C NMR studies, which indicate that E95A forms an acetyl-S-enzyme reaction intermediate with the same distinctive spectroscopic characteristics measured using wild type enzyme. The initial reaction steps are not disrupted in E95A, which exhibits normal levels of Michaelis complex and acetyl-S-enzyme intermediate. Likewise, E95A is not impaired in catalysis of the terminal reaction step, as indicated by efficient catalysis of a hydrolysis partial reaction. Single turnover experiments indicate defective C-C bond formation. The mechanism-based inhibitor, 3-chloropropionyl-CoA, efficiently alkylates E95A. This is compatible with the presence of a functional general base, raising the possibility that Glu(95) functions as a general acid. Demonstration of a significant upfield shift for the methyl protons of HMG-CoA synthase's acetyl-S-enzyme reaction intermediate suggests a hydrophobic active site environment that could elevate the pK(a) of Glu(95) as required to support its function as a general acid.
Asunto(s)
Ácido Glutámico/metabolismo , Hidroximetilglutaril-CoA Sintasa/metabolismo , Acilcoenzima A/metabolismo , Alquilación , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Catálisis , Hidroximetilglutaril-CoA Sintasa/genética , Espectroscopía de Resonancia Magnética , Modelos Químicos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Alineación de Secuencia , Relación Estructura-ActividadRESUMEN
The effect of nonhydrolyzable guanine nucleotides on mammalian acetyl CoA carboxylase (ACC) activity was examined. Using porous rat adipocytes and crude fat cell homogenates to study metabolic pathway flux, GMPPNP and/or GTP gamma S inhibited [14C]fatty acid formation by up to 95% when either [6-14C]glucose-6-phosphate or [1-14C]acetyl CoA was used as substrate. If [2-14C]malonyl CoA initiated flux, however, no inhibition was apparent. These pathway flux studies suggested that ACC was the locus of inhibition, and that the mechanism might involve a disruption of guanine nucleotide hydrolysis by the nonhydrolyzable analogues. Using partially and avidin-sepharose-purified ACC preparations from rat fat, liver and mammary tissue, citrate-stimulated ACC activity was inhibited by 25-75% with 50 microM GTP gamma S. Related compounds and nucleotides had absent-to-minimal effects on ACC. ATP gamma S was inhibitory (10-30% at 5-15 microM), but always to a lesser degree than equimolar GTP gamma S. Filter binding assays with [alpha-32P]GTP or [35S]GTP gamma S were negative, but low-level GTPase activity was detected. Using photoaffinity labelling techniques, [alpha-32P]GTP was found to bind ACC and not pyruvate carboxylase. The hypothesis that citrate-responsive ACC activity may be modulated by an intrinsic or associated GTP binding site is explored. Since ACC forms polymers, as does the cytoskeletal protein beta-tubulin, amino acid sequence comparisons between ACC and atypical GTP binding domain of beta tubulin are presented.