RESUMEN
The oxidation of (2'S)-nicotine in the active site of human cytochrome P450 2A6 has been subjected to a detailed analysis by theoretical quantum mechanical/molecular mechanical (QM/MM) calculations linked with a theoretical and experimental study of the associated isotope effects. The study has focused on seeking an explanation as to why oxidation at the 5'-carbon position (A) is favored over oxidation at the methyl carbon (CMe) position (B). It is deduced that the choice of hydrogen for abstraction is not determined by geometric features of the active site, but by the lower energy barrier associated with 5' oxidation. N-Demethylation leading to N-hydroxymethylnornicotine requires ca. 6.5 kcal/mol more energy to transfer a hydrogen atom than is required for oxidation on the carbon 5'. Neither protonation of the pyrrolidine nitrogen (N1') nor inclusion of a water molecule in the reaction process influences the balance between the two oxidation pathways. In both cases, the hydrogen transfer step is rate limiting. An analysis of the calculated kinetic isotope effects indicates that the presence of a (2)H in either the C5' or the CMepositions has a significant effect on the reaction kinetics. However, the experimental values of around 2.2-2.6 are considerably lower than those predicted by theoretical calculations (9.3 and 6.9 for C5' or the CMe positions, respectively, in the LS state of Cpd I), typical of the masking commonly found for CYP450 reactions. The fact that similar values are found for cotinine formation from both substrates, however, may indicate that the measured value is not that for H-abstraction but, rather, is a combined value for (2)H influence on electronic redistribution between iminium states of the pyrrolidine ring. This is the first time that oxidation at the C5' or the CMe positions has been directly compared and that isotope effects have been obtained for this reaction in a human cytochrome P450 reaction.
Asunto(s)
Carbono/química , Sistema Enzimático del Citocromo P-450/metabolismo , Nicotina/metabolismo , Biocatálisis , Humanos , Hidroxilación , Modelos Moleculares , Simulación de Dinámica Molecular , Estructura Molecular , Nicotina/química , Oxidación-Reducción , Teoría CuánticaRESUMEN
(15)N heavy isotope effects are especially useful when detail is sought pertaining to the reaction mechanism for the cleavage of a C-N bond. Their potential in assisting to describe the mechanism of N-demethylation of tertiary amines by the action of cytochrome P450 monooxygenase has been investigated. As a working model for the first step, oxidation of the N-methyl group to N-methoxyl, tropine and a cytochrome P450 monooxygenase reaction centre composed of a truncated heme with sulfhydryl as the axial ligand were used. It is apparent that this first step of the reaction proceeds via a hydrogen atom transfer mechanism. Transition states for this step are described for both the high spin ((4)TS(H)) and low spin ((2)TS(H)) pathways in both gas and solvation states. Hence, overall normal secondary (15)N KIE could be calculated for the reaction path modeled in the low spin state, and inverse for the reaction modeled in the high spin state. This partial reaction has been identified as the probable rate limiting step. The model for the second step, fission of the C-N bond, consisted of N-methoxylnortropine and two molecules of water. A transition state described for this step, TS(CN), gives a strongly inverse overall theoretical (15)N KIE.
Asunto(s)
Aminas/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Pseudomonas putida/enzimología , Aminas/química , Sistema Enzimático del Citocromo P-450/química , Electrones , Hidroxilación , Metilación , Modelos Moleculares , Isótopos de Nitrógeno/metabolismo , Teoría Cuántica , Tropanos/química , Tropanos/metabolismoRESUMEN
N-Demethylation of tropine is an important step in the degradation of this compound and related metabolites. With the purpose of understanding the reaction mechanism(s) involved, it is desirable to measure the 15N kinetic isotope effects (KIEs), which can be accessed through the 15N isotope shift (Deltadelta15N) during the reaction. To measure the isotope fractionation in 15N during tropine degradation necessitates the extraction of the residual substrate from dilute aqueous solution without introducing artefactual isotope fractionation. Three protocols have been compared for the extraction and measurement of the 15N/14N ratio of tropine from aqueous medium, involving liquid-liquid phase partitioning or silica-C18 solid-phase extraction. Quantification was by gas chromatography (GC) on the recovered organic phase and delta15N values were obtained by isotope ratio measurement mass spectrometry (irm-MS). Although all the protocols used can provide satisfactory data and both irm-EA-MS and irm-GC-MS can be used to obtain the delta15N values, the most convenient method is liquid-liquid extraction from a reduced aqueous volume combined with irm-GC-MS. The protocols are applied to the measurement of 15N isotope shifts during growth of a Pseudomonas strain that uses tropane alkaloids as sole source of carbon and nitrogen. The accuracy of the determination of the 15N/14N ratio is sufficient to be used for the determination of 15N-KIEs.
Asunto(s)
Fraccionamiento Químico/métodos , Isótopos de Nitrógeno/química , Tropanos/química , Espectrometría de Masas/métodos , Peso Molecular , Nitrógeno/química , Nitrógeno/metabolismo , Isótopos de Nitrógeno/metabolismo , Pseudomonas/química , Pseudomonas/crecimiento & desarrollo , Pseudomonas/metabolismo , Tropanos/metabolismoRESUMEN
Isotope fractionation is a powerful technique by which to probe the reaction mechanism of enzymes. The effect of a heavy isotope on the reaction energetics can be used to predict transition state architecture and reaction mechanism. In order to examine simultaneously the isotope fractionation in (13)C at multiple sites within the substrate and product molecules without any need for site-selective isotope enrichment, a technique exploiting quantitative isotopic nuclear magnetic resonance (NMR) spectrometry at natural abundance (NAQ-NMR) has been developed. Here we report the first application of this technique to the study of an enzyme-catalyzed reaction, the bioconversion of ferulic acid to vanillin in cultures of Streptomyces setonii. We were able to show that the NAQ-NMR methodology is sufficiently precise and robust to measure the isotope shifts in the (13)C/(12)C ratios in both substrate and product of this biotransformation, thereby permitting meaningful data to be obtained even at carbon positions that take part only indirectly in the reaction and show only secondary isotope fractionation. The results obtained provide direct evidence in support of the current hypothesis for the reaction mechanism of the enzyme hydroxycinnamoyl-CoA hydratase/lyase, notably the proposed involvement of the quinone methide enolate of feruloyl-CoA as intermediate in the catalytic pathway.
Asunto(s)
Benzaldehídos/metabolismo , Isótopos de Carbono/análisis , Ácidos Cumáricos/metabolismo , Espectroscopía de Resonancia Magnética/métodos , Métodos Analíticos de la Preparación de la Muestra , Benzaldehídos/química , Benzaldehídos/aislamiento & purificación , Ácidos Cumáricos/química , Ácidos Cumáricos/aislamiento & purificación , Hidroliasas/metabolismo , Streptomyces/enzimologíaRESUMEN
Different members of the alcohol oxidoreductase family can transfer the hydride of NAD(P)H to either the re- or the si-face of the substrate. The enantioselectivity of transfer is very variable, even for a range of substrates reduced by the same enzyme. Exploiting quantitative isotopic (2)H NMR to measure the transfer of (2)H from NAD(P)(2)H to ethanol, a range of enantiomeric excess between 0.38 and 0.98, depending on the origin of the enzyme and the nature of the cofactor, has been determined. Critically, in no case was only (R)-[1-(2)H]ethanol or (S)-[1-(2)H]ethanol obtained. By calculating the relative energies of the active site models for hydride transfer to the re- or si-face of short-chain aldehydes by alcohol dehydrogenase from Saccharomyces cerevisiae and Lactobacillus brevis, it is shown that the differences in the energy of the systems when the substrate is positioned with the alkyl group in one or the other pocket of the active site could play a role in determining stereoselectivity. These experiments help to provide insight into structural features that influence the potential catalytic flexibility of different alcohol dehydrogenase activities.
Asunto(s)
Butiril-CoA Deshidrogenasa/química , Butiril-CoA Deshidrogenasa/metabolismo , Aminoácidos/análisis , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Calorimetría , Dominio Catalítico , Etanol/metabolismo , Cinética , Levilactobacillus brevis/enzimología , Espectroscopía de Resonancia Magnética/métodos , Modelos Moleculares , NAD/metabolismo , NADP/metabolismo , Conformación Proteica , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidad por Sustrato , TermodinámicaRESUMEN
Heavy-atom isotope effects for the N-demethylation of nicotine have been determined in vivo in static-phase biosynthetically incompetent plant cell cultures of Nicotiana species. A (2)H kinetic isotope effect of 0.587 and a (15)N kinetic isotope effect of 1.0028 were obtained. An identical (15)N kinetic isotope effect of 1.0032 was obtained for the nicotine analogue, N-methyl-2-phenylpyrrolidine. The magnitude of the (15)N heavy-atom isotope effect indicates that the fission of the CN bond is not rate limiting for demethylation. The theoretical calculation of heavy-atom isotope effects for a model of the reaction pathway based on cytochrome P450 best fits the measured kinetic isotope effect to the addition of hydroxyl ion to iminium to form N-hydroxymethyl, for which the computed (2)H- and (15)N kinetic isotope effects are 0.689 and 1.0081, respectively. This large inverse (2)H kinetic isotope effect is not compatible with the initial abstraction of the H from the methyl group playing a significant kinetic role in the overall kinetic limitation of the reaction pathway, since computed values for this step (4.54 and 0.9995, respectively) are inconsistent with the experimental data.
Asunto(s)
Hidróxidos/metabolismo , NADPH-Ferrihemoproteína Reductasa/metabolismo , Nicotiana/metabolismo , Nicotina/metabolismo , Deuterio/metabolismo , Metilación , Modelos Químicos , Isótopos de Nitrógeno/metabolismo , Pirrolidinas/metabolismo , Nicotiana/enzimologíaRESUMEN
We report the influence of the substituent at the N atom of the ligands on the synthesis, biological activity, and stability of Pd(II) complexes of the general formula PdL(2). The compounds adopt a cis or trans configuration with respect to the substituent at the nitrogen atom. Sterically hindered substituents promote the formation of trans isomers, whereas when the nitrogen atom is unsubstituted, cis isomers are formed. The compounds were characterized by elemental analysis, infrared and 1H NMR spectroscopies, and electrospray mass spectrometry. The complexes were also studied using X-ray diffraction and computational DFT methods. Both complexes cis-3a and trans-3c exhibit square-planar geometries around the Pd(II) atom. The cytotoxic effects of these complexes were examined on two human leukemia cell lines, HL-60 and NALM-6. Pd complex cis-3a showed significant cytotoxic activity. The effects exhibited by this complex were comparable to those reported for carboplatin. Loigand 2a was not cytotoxic. Computational analysis carried out at the PB/B3LYP/LACVP**//mPW1PW91/LanL2DZ level showed excellent correlation between the energy difference of the cis and trans isomers and the cytotoxic activity, rendering computations a useful predictive tool for the design of new drugs.
Asunto(s)
Cumarinas/química , Compuestos Organoplatinos/química , Compuestos Organoplatinos/farmacología , Antineoplásicos/química , Antineoplásicos/farmacología , Células Cultivadas , Cristalografía por Rayos X , Ensayos de Selección de Medicamentos Antitumorales , Células HL-60 , Humanos , Espectroscopía de Resonancia Magnética , Modelos Químicos , Modelos Moleculares , Estructura Molecular , Nitrógeno/química , Compuestos Organoplatinos/síntesis química , Teoría Cuántica , Relación Estructura-ActividadRESUMEN
dl-2-Haloacid dehalogenase from Pseudomonas sp. 113 is a unique enzyme because it acts on the chiral carbons of both enantiomers, although its amino acid sequence is similar only to that of d-2-haloacid dehalogenase from Pseudomonas putida AJ1 that specifically acts on (R)-(+)-2-haloalkanoic acids. Furthermore, the catalyzed dehalogenation proceeds without formation of an ester intermediate; instead, a water molecule directly attacks the alpha-carbon of the 2-haloalkanoic acid. We have studied solvent deuterium and chlorine kinetic isotope effects for both stereoisomeric reactants. We have found that chlorine kinetic isotope effects are different: 1.0105 +/- 0.0001 for (S)-(-)-2-chloropropionate and 1.0082 +/- 0.0005 for the (R)-(+)-isomer. Together with solvent deuterium isotope effects on V(max)/K(M), 0.78 +/- 0.09 for (S)-(-)-2-chloropropionate and 0.90 +/- 0.13 for the (R)-(+)-isomer, these values indicate that in the case of the (R)-(+)-reactant another step preceding the dehalogenation is partly rate-limiting. Under the V(max) conditions, the corresponding solvent deuterium isotope effects are 1.48 +/- 0.10 and 0.87 +/- 0.27, respectively. These results indicate that the overall reaction rates are controlled by different steps in the catalysis of (S)-(-)- and (R)-(+)-reactants.
Asunto(s)
Hidrolasas/metabolismo , Hidrolasas/química , Cinética , Modelos Moleculares , Espectrometría de Masa Bombardeada por Átomos Veloces , Espectrofotometría UltravioletaRESUMEN
ONIOM calculations have provided novel insights into the mechanism of homolytic Co-C5' bond cleavage in the 5'-deoxyadenosylcobalamin cofactor catalyzed by methylmalonyl-CoA mutase. We have shown that it is a stepwise process in which conformational changes in the 5'-deoxyadenosine moiety precede the actual homolysis step. In the transition state structure for homolysis, the Co-C5' bond elongates by approximately 0.5 Angstroms from the value found in the substrate-bound reactant complex. The overall barrier to homolysis is approximately 10 kcal/mol, and the radical products are approximately 2.5 kcal/mol less stable than the initial ternary complex of enzyme, substrate, and cofactor. The movement of the deoxyadenosine moiety during the homolysis step positions the resulting 5'-deoxyadenosyl radical for the subsequent hydrogen atom transfer from the substrate, methylmalonyl-CoA.
Asunto(s)
Cobamidas/química , Metilmalonil-CoA Mutasa/química , Adenosina/química , Adenosina/metabolismo , Sitios de Unión , Cobamidas/metabolismo , Enlace de Hidrógeno , Metilmalonil-CoA Mutasa/metabolismo , Modelos Moleculares , Conformación Proteica , TermodinámicaRESUMEN
Several semiempirical methods (MNDO-d, PM3tm, PM3-d, PM5, PM6, and AM1-d) have been tested against experimental data and density functional theory (DFT) results in search for the best methods that can be used for quantum-mechanical-molecular mechanics (QM/MM) modeling of corrinoid systems of vitamin B(12) co-factor. It has been found that the PM6 parametrization in its present form gives results closest to hybrid DFT calculations that are most widely used thus far. In comparison with pure DFT and experimental data the best agreement is obtained for PM3tm parametrization, while PM6 yields slightly worse results. AM1-d yields bad geometry of the corrin moiety. The worst performance was observed for MNDO-d, which has severe problem with position and orientation of the alpha-ligands.