RESUMO
D-hydantoinase from Vigna angularis was immobilized by covalent linkage to aminopropyl glass beads. Thermal stability, resistance to storage at different pH values and temperatures of this biocatalyst were studied. This enzyme preparation was used as a catalyst to prepare enantioenriched N-carbamoyl-D-phenylglycine, N-carbamyl-D-p-fluorophenylglycine and N-carbamoyl-D-p-trifluoromethylphenylglycine, using a stirred batch reactor. Reactions were conducted during eight repeated reaction cycles, without loss of enzymatic activity or variation of the enantiomeric excess of the respective product (>98%).
Assuntos
Amidoidrolases/química , Enzimas Imobilizadas , Glicina/química , Extratos Vegetais , Proteínas de Plantas/química , Plantas/enzimologia , Reatores Biológicos , Catálise , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Modelos Moleculares , Estereoisomerismo , Temperatura , Fatores de TempoRESUMO
1The Enantiomeric Ratio (E) of the enzyme, acting as specific catalysts in resolution of enantiomers, is an important parameter in the quantitative description of these chiral resolution processes. In the present work, two novel methods hereby called Method I and II, for estimating E and the kinetic parameters Km and Vm of enantiomers were developed. These methods are based upon initial rate (v) measurements using different concentrations of enantiomeric mixtures (C) with several molar fractions of the substrate (x). Both methods were tested using simulated "experimental data" and actual experimental data. Method I is easier to use than Method II but requires that one of the enantiomers is available in pure form. Method II, besides not requiring the enantiomers in pure form shown better results, as indicated by the magnitude of the standard errors of estimates. The theoretical predictions were experimentally confirmed by using the oxidation of 2-butanol and 2-pentanol catalyzed by Thermoanaerobium brockii alcohol dehydrogenase as reaction models. The parameters E, Km and Vm were estimated by Methods I and II with precision and were not significantly different from those obtained experimentally by direct estimation of E from the kinetic parameters of each enantiomer available in pure form.
RESUMO
D-Hydantoinase from Vigna angularis hydrolyzed rac-5-monosubstituted-hydantoins with polar and aromatic side chains and dihydrothymine but rac-5,5-disubstituted-hydantoins were not substrates of this enzyme. 5-Phenylhydantoin was the best substrate. By using this substrate, Ncarbamoyl-D-phenylglycine was obtained in quantitative yield and over 98% ee.
Assuntos
Amidoidrolases/metabolismo , Hidantoínas/metabolismo , Fenilacetatos/metabolismo , Rosales/metabolismo , Ureia/análogos & derivados , Ureia/metabolismo , Hidrólise , Conformação Molecular , Rosales/química , Rosales/enzimologia , Especificidade por SubstratoRESUMO
1. The reaction of reduction of oxaloacetate to L-malate in the presence of NADH catalyzed by mitochondrial malate dehydrogenase (EC 1.1.1.37) of Toxocara canis muscle has been studied. 2. The data obtained in initial velocity experiments as well as those involving product inhibition suggest that the reaction mechanism is of the sequential type with a kinetically significant ternary complex and in which the coenzymes bind to the free enzyme. 3. The kinetic parameters, including the inhibition constant for NADH were estimated by non-linear regression analysis using the appropriate rate equations.
Assuntos
Malato Desidrogenase/metabolismo , Mitocôndrias Musculares/enzimologia , Oxaloacetatos/metabolismo , Toxocara/enzimologia , Animais , Cães , Cinética , Malatos/metabolismo , NAD/metabolismo , OxirreduçãoRESUMO
The activity of horse liver alcohol dehydrogenase was inhibited by phenylhydrazine. Kinetic experiments showed that this compound produced linear competitive inhibition with respect to NAD+ and linear noncompetitive inhibition with respect to ethanol. These results suggested that the inhibitor competes with NAD+ for the coenzyme binding site of alcohol dehydrogenase, forming a dead-end complex with the free form of the enzyme. A Ki value of 393 +/- 51 microM was estimated for the enzyme-inhibitor complex. Further evidence for this mechanism of inhibition arose from the fact that the same kind of inhibition was found for rabbit muscle lactate dehydrogenase. The Ki value for the lactate dehydrogenase-phenylhydrazine complex was 43.41 +/- 2.10 mM. The significant difference between these Ki values is explained in terms of known differences in hydrophobicity of the nicotinamide binding region in the two enzymes.
Assuntos
Álcool Desidrogenase/antagonistas & inibidores , L-Lactato Desidrogenase/antagonistas & inibidores , Fígado/enzimologia , Músculos/enzimologia , Fenil-Hidrazinas/farmacologia , Animais , Etanol/metabolismo , Cavalos , Hidrazinas/farmacologia , Cinética , NAD/metabolismo , Coelhos , Semicarbazidas/farmacologiaRESUMO
Purified mitochondrial malate dehydrogenase isoenzyme (m-MDH) of Toxocara canis muscle presented maximum activity at 48 degrees C. A clear change in slope of the Arrhenius plot was observed. The energy of activation calculated for the catalytic process showed values of 3.2 kcal/mol and 10.5 kcal/mol. Thermal inactivation of m-MDH showed that it is more thermolabile than the s-isoenzyme. The inactivation of the enzyme by heat could be reduced at least in part by the addition of 0.1 mM NADH. The heat denaturation showed to be a first-order process. The rate constant (k) was calculated as being of the order of 5.28 X 10(-4) s-1 at 40 degrees C. The activation energy for the heat inactivation process was 16.45 kcal/mol between 30 degrees C and 40 degrees C and 13.79 kcal/mol between 40 degrees C and 48 degrees C.
Assuntos
Malato Desidrogenase/metabolismo , Toxocara/enzimologia , Animais , Isoenzimas/metabolismo , Cinética , Mitocôndrias Musculares/enzimologia , Desnaturação Proteica , Temperatura , TermodinâmicaRESUMO
This paper describes a microcomputer program written in BASIC for fitting different two-substrate enzyme kinetic mechanisms. The present program is a complement of one which was recently published and was developed to fit different models of enzyme inhibition. The complete program resulting from this complementation allows the fitting of the most usual rate equations found in steady-state studies of enzyme kinetics. The program based on a non-linear least-squares regression, can be run on any microcomputer having the CP/M operating system. The initial rate equation for a steady-state random bisubstrate mechanism was chosen to evaluate the performance of the program, with contrived data of known error structure.
Assuntos
Computadores , Enzimas/metabolismo , Microcomputadores , Software , Cinética , Modelos Biológicos , Design de SoftwareRESUMO
A specific deficiency in UDPG-linked trehalose-6-phosphate synthase in the yeast, Saccharomyces cerevisiae has been associated with a single nuclear gene, sst1. Strains bearing this abnormal allele lacked the capacity to accumulate trehalose during growth on glucose or galactose medium or when incubated with glucose in nonproliferating conditions. However, sst1 strains still exhibited trehalose accumulation during growth on maltose medium, provided they contained a gene for maltose fermentation (MAL gene). Introduction of a constitutive MAL (c) gene into an sst1 strain rendered the strain capable of accumulating trehalose during growth on glucose medium, but did not restore the normal capacity to convert glucose to trehalose in nonproliferating conditions. Different systems, I and II, of trehalose accumulation are proposed. System I would require the UPDG-linked synthase, whereas system II, which is normally specific for maltose, would utilize a different enzyme. It is unlikely that system II produces trehalose by trans-glucosylation, since it converted glucose to trehalose in MAL (c) sst1 strains. The results indicate that maltose specifically induces the production of the MAL gene-product, which, in turn, would stimulate the formation (or activation) of system II.