Stabilisation of methylene radicals by cob(II)alamin in coenzyme B12 dependent mutases

Coenzyme B12 initiates radical chemistry in two types of enzymatic reactions, the irreversible eliminases (e.g., diol dehydratases) and the reversible mutases (e.g., methylmalonyl-CoA mutase). Whereas eliminases that use radical generators other than coenzyme B12 are known, no alternative coenzyme B12 independent mutases have been detected for substrates in which a methyl group is reversibly converted to a methylene radical. We predict that such mutases do not exist. However, coenzyme B12 independent pathways have been detected that circumvent the need for glutamate, beta-lysine or methylmalonyl-CoA mutases by proceeding via different intermediates. In humans the methylcitrate cycle, which is ostensibly an alternative to the coenzyme B12 dependent methylmalonyl-CoA pathway for propionate oxidation, is not used because it would interfere with the Krebs cycle and thereby compromise the high-energy requirement of the nervous system. In the diol dehydratases the 5'-deoxyadenosyl radical generated by homolysis of the carbon-cobalt bond of coenzyme B12 moves about 10 A away from the cobalt atom in cob(II)alamin. The substrate and product radicals are generated at a similar distance from cob(II)alamin, which acts solely as spectator of the catalysis. In glutamate and methylmalonyl-CoA mutases the 5'-deoxyadenosyl radical remains within 3-4 A of the cobalt atom, with the substrate and product radicals approximately 3 A further away. It is suggested that cob(II)alamin acts as a conductor by stabilising both the 5'-deoxyadenosyl radical and the product-related methylene radicals.     

B12-retro-riboswitches: guanosyl-induced constitutional switching of B12 coenzymes

Complete B12 derivatives are natural "molecular switches" as a result of the coordinative switch ("base on" or "base off") of the natural nucleotide base. Certain predesigned B12-nucleotide conjugates were shown recently to behave as "retro riboswitches", in which the nucleotide environment modified the equilibrium between these two isomeric B12 structures. In contrast, the "reverse" situation has been discovered in natural B12 riboswitches, in which the binding of coenzyme B12 induces a conformational switch in the RNA species. The first (predesigned) B12-retro-riboswitches were DNA conjugates of methylcobalamin. We describe herein two representative B12-retro-riboswitches, in which an appended (RNA) nucleotide is used to destabilize the base-on form and induce the base-on to base-off switch. Through use of heterogeneous solid-phase synthetic methods, Co(beta)-cyanocobalamin-(3'-->2')-2'-methoxyguaninyl-3'-ate was prepared first as the crucial covalent RNA conjugate of vitamin B12. This cyanocorrinoid opened the door to two organometallic B12-nucleotide conjugates, which were made by electrosynthetic means: the cyanocorrinoid was cleanly methylated or adenosylated at the cobalt center to furnish covalent RNA conjugates of the organometallic B12 cofactors methylcobalamin and coenzyme B12, respectively. At room temperature, aqueous solutions of both of these organometallic RNA-B12 conjugates exhibited properties indicative of significant weakening of the axial (Co--N) bond (of their base-on forms) and of an enhanced formation of the base-off species. The base-on to base-off switch was studied by UV/Vis and NMR spectroscopic studies, which showed that the switch was very temperature-dependent and was accentuated with increasing temperatures. Thermodynamic data of the two organometallic RNA-B12 conjugates revealed an important contribution of entropic effects to the observed base-on to base-off switch. The two organometallic RNA-B12 conjugates thus acted as B12-retro-riboswitches and allowed the observation of a temperature-dependent reverse switch in the B12 cofactor moiety, induced by the appended nucleotide moiety. This behavior may be of interest in the "RNA-world" hypothesis, in which (simple) B12 derivatives are thought to act as possible catalytic enhancers ("cofactors") in RNA-based "B12 ribozymes".     

Homocoenzyme B12 and bishomocoenzyme B12: covalent structural mimics for homolyzed, enzyme-bound coenzyme B12

Efficient electrochemical syntheses of "homocoenzyme B(12)" (2, Co(beta)-(5'-deoxy-5'-adenosyl-methyl)-cob(III)alamin) and "bishomocoenzyme B(12)" (3, Co(beta)-[2-(5'-deoxy-5'-adenosyl)-ethyl]-cob(III)alamin) are reported here. These syntheses have provided crystalline samples of 2 and 3 in 94 and 77 % yield, respectively. In addition, in-depth investigations of the structures of 2 and 3 in solution were carried out and a high-resolution crystal structure of 2 was obtained. The two homologues of coenzyme B(12) (2 and 3) are suggested to function as covalent structural mimics of the hypothetical enzyme-bound "activated" (that is, "stretched" or even homolytically cleaved) states of the B(12) cofactor. From crude molecular models, the crucial distances from the corrin-bound cobalt center to the C5' atom of the (homo)adenosine moieties in 2 and 3 were estimated to be about 3.0 and 4.4 A, respectively. These values are roughly the same as those found in the two "activated" forms of coenzyme B(12) in the crystal structure of glutamate mutase. Indeed, in the crystal structure of 2, the cobalt center was observed to be at a distance of 2.99 A from the C5' atom of the homoadenosine moiety and the latter was found to be present in the unusual syn conformation. In solution, the organometallic moieties of 2 and 3 were shown to be rather flexible and to be considerably more dynamic than the equivalent group in coenzyme B(12). The homoadenosine moiety of 2 was indicated to occur in both the syn and the anti conformations.     

Simultaneous determination of vitamin B12 and its derivatives using some of multivariate calibration 1 (MVC1) techniques

Resolution of binary mixtures of vitamin B12, methylcobalamin and B12 coenzyme with minimum sample pre-treatment and without analyte separation has been successfully achieved by methods of partial least squares algorithm with one dependent variable (PLS1), orthogonal signal correction/partial least squares (OSC/PLS), principal component regression (PCR) and hybrid linear analysis (HLA). Data of analysis were obtained from UV-vis spectra. The UV-vis spectra of the vitamin B12, methylcobalamin and B12 coenzyme were recorded in the same spectral conditions. The method of central composite design was used in the ranges of 10-80mgL(-1) for vitamin B12 and methylcobalamin and 20-130mgL(-1) for B12 coenzyme. The models refinement procedure and validation were performed by cross-validation. The minimum root mean square error of prediction (RMSEP) was 2.26mgL(-1) for vitamin B12 with PLS1, 1.33mgL(-1) for methylcobalamin with OSC/PLS and 3.24mgL(-1) for B12 coenzyme with HLA techniques. Figures of merit such as selectivity, sensitivity, analytical sensitivity and LOD were determined for three compounds. The procedure was successfully applied to simultaneous determination of three compounds in synthetic mixtures and in a pharmaceutical formulation.     

Synthesis, spectroscopic characterization, axial base coordination equilibrium and photolytic kinetics studies of a new coenzyme B12 analogue-3'-deoxy-2',3'-anhydrothymidylcobalamin

A new coenzyme B12 (AdoCbl) analogue, 3'-deoxy-2',3'-didehydrothymidylcobalamin (2',3'-anThyCbl) was prepared by the reaction of 5'-iodo-3'-deoxy-2',3'-dihydrothmidine with reduced B12a, and characterized by UV-Vis, CD, ESI-MS and NMR spectroscopies. Its axial base (dbzm) coordination equilibria with pH's and temperatures were investigated and showed similar features to those of coenzyme B12. Photolytic dynamics studies under homolytic and heterolytic conditions demonstrated that the Co-C bond of the analogue is slightly more photolabile relative to coenzyme B12.     

High-performance liquid chromatography-fluorometry for the determination of thiols in biological samples using N-[4-(6-dimethylamino-2-benzofuranyl) phenyl]-maleimide

The selective determination of thiols in biological samples was investigated by high-performance liquid chromatography using N-[4-(6-dimethylamino-2-benzofuranyl)phenyl] maleimide, which was found to give fluorescent products when treated with certain thiols. Six kinds of thiol (reduced glutathione, cysteine, N-acetylcysteine, cysteamine, homocysteine and coenzyme A) could be separated simultaneously within ca. 12 min and determined at final level of sensitivity. The method was successfully applied to the determination of thiols in rat tissues and plasma and in human normal serum.     

A combined density functional theory and molecular mechanics study of the relationship between the structure of coenzyme B12 and its binding to methylmalonyl-CoA mutase

A combined density functional theory (DFT) and molecular mechanics (MM) approach was applied to investigate the relationship between the structure of a free coenzyme B12, and bound to methylmalonyl-CoA mutase. It was found that, upon coenzyme binding to apoenzyme, the Co-C bond remains intact, while the C-Naxial bond becomes slightly elongated and labilized. The labilization of the Co-Naxial bond that takes place in coenzyme B12-dependent enzymes is most likely necessary for fine-tuning of the cobalt-nitrogen (axial base) distance. The controlling of this distance is important to inhibit abiological site reaction involving heterolysis of the Co-C bond but is not important for biologically relevant Co-C bond homolysis.     

NMR observations of 13C-enriched coenzyme B12 bound to the ribonucleotide reductase from Lactobacillus leichmannii

The 13C NMR resonance and one-bond 1H-13C coupling constants of coenzyme B12 enriched in 13C in the cobalt-bound carbon have been observed in the complex of the coenzyme with the B12-dependent ribonucleotide reductase from Lactobacillus leichmannii. Neither the 13C NMR chemical shift nor the 1H-13C coupling constants are significantly altered by binding of the coenzyme to the enzyme. The results suggest that ground-state Co-C bond distortion is not utilized by this enzyme to activate coenzyme B12 for C-Co bond homolysis.     

Polynucleotide analogues. VI. Synthesis and characterization of alternating copolymers of maleic anhydride and dihydropyran containing guanine derivatives

2-Amino-6-chloropurine was reacted with 2-(tosyloxymethyl)-2,3-dihydro-2H-pyran to give 2-(2-amino-6-chloropurin-9-ylmethyl)-2,3-dihydro-2H-pyran ( 3 ) and its N⁷-isomer ( 4 ), which were treated with 5% aqueous trimethylamine to result in 2-(guanin-9-ylmethyl)-2,3-dihydro-2H-pyran ( 5 ) and its N⁷-isomer ( 6 ), respectively. 2-(N²-Acetylguanin-9-yl-methyl)-3,4-dihydro-2H-pyran ( 7 ) and 2-(N²-acetylguanin-7-ylmethyl)-3,4-dihydro-2H-pyran ( 8 ), obtained by acetylation of compounds 5 and 6 , were copolymerized with maleic anhydride to give the alternating copolymers 9 and 10 , and they were hydrolyzed to result in poly[ {2-(guanin-9-ylmethyl)tetrahydropyran-5,6-diyl} {1,2-dicarboxyethylene}] ( 11 ) and poly[ {2-(guanin-7-ylmethyl)tetrahydropyran-5,6-diyl} {1,2-dicarboxyethylene}] ( 12 ), re-spectively. Polymer 11 showed hypochromicity whereas 12 exhibited hyperchromicity in aqueous solutions. Polymers 11 and 12 in aqueous solutions showed very strong excimer fluorescence with the maximum intensities at 432 and 446 nm, respectively, at room tem-perature. The two polymers showed polyelectrolyte effects, e.g., very high GPC molecular weights as well as reduced viscosities at low concentrations in water. Normal behavior was retained by addition of inorganic salts. Sodium salts of polymers 11 and 12 migrated to the anode by electrophoresis and both showed two bands. © 1995 John Wiley & Sons, Inc.     

The enzymatic activation of coenzyme B12

The enzymatic "activation" of coenzyme B12 (5'-deoxyadenosylcobalamin, AdoCbl), in which homolysis of the carbon-cobalt bond of the coenzyme is catalyzed by some 10(9)- to 10(14)-fold, remains one of the outstanding problems in bioinorganic chemistry. Mechanisms which feature the enzymatic manipulation of the axial Co-N bond length have been investigated by theoretical and experimental methods. Classical mechanochemical triggering, in which steric compression of the long axial Co-N bond leads to increased upward folding of the corrin ring and stretching of the Co-C bond is found to be feasible by molecular modeling, but the strain induced in the Co-C bond seems to be too small to account for the observed catalytic power. The modeling study shows that the effect is a steric one which depends on the size of the axial nucleotide base, as substitution of imidazole (Im) for the normal 5,6-dimethylbenzimidazole (Bzm) axial base decreases the Co-C bond labilization considerably. An experimental test was thus devised using the coenzyme analog with Im in place of Bzm (Ado(Im)Cbl). Studies of the enzymatic activation of this analog by the B12-dependent ribonucleoside triphosphate reductase from Lactobacillus leichmannii coupled with studies of the non-enzymatic homolytic lability of the Co-C bond of Ado(Im)Cbl show that the enzyme is only slightly less efficient (3.8-fold, 0.8 kcal mol(-1)) at activating Ado(Im)Cbl than at activating AdoCbl itself. This suggests, in agreement with the modeling study, that mechanochemical triggering can make only a small contribution to the enzymatic activation of AdoCbl. Another possibility, electronic stabilization of the Co(II) homolysis product by compression of the axial Co-N bond, requires that enzymatic activation be sensitive to the basicity of the axial nucleotide. Preliminary studies of the enzymatic activation of a coenzyme analog with a 5-fluoroimidazole axial nucleotide suggest that the catalysis of Co-C bond homolysis may indeed be significantly slowed by the decrease in basicity.     

Acid-, base-, and lewis-acid-catalyzed heterolysis of methoxide from an alpha-hydroxy-beta-methoxy radical: models for reactions catalyzed by coenzyme B12-dependent diol dehydratase

[Reaction: see text].A model for glycol radicals was employed in laser flash photolysis kinetic studies of catalysis of the fragmentation of a methoxy group adjacent to an alpha-hydroxy radical center. Photolysis of a phenylselenylmethylcyclopropane precursor gave a cyclopropylcarbinyl radical that rapidly ring opened to the target alpha-hydroxy-beta-methoxy radical (3). Heterolysis of the methoxy group in 3 gave an enolyl radical (4a) or an enol ether radical cation (4b), depending upon pH. Radicals 4 contain a 2,2-diphenylcyclopropane reporter group, and they rapidly opened to give UV-observable diphenylalkyl radicals as the final products. No heterolysis was observed for radical 3 under neutral conditions. In basic aqueous acetonitrile solutions, specific base catalysis of the heterolysis was observed; the pK(a) of radical 3 was determined to be 12.5 from kinetic titration plots, and the ketyl radical formed by deprotonation of 3 eliminated methoxide with a rate constant of 5 x 10(7) s(-1). In the presence of carboxylic acids in acetonitrile solutions, radical 3 eliminated methanol in a general acid-catalyzed reaction, and rate constants for protonation of the methoxy group in 3 by several acids were measured. Radical 3 also reacted by fragmentation of methoxide in Lewis-acid-catalyzed heterolysis reactions; ZnBr2, Sc(OTf)3, and BF3 were found to be efficient catalysts. Catalytic rate constants for the heterolysis reactions were in the range of 3 x 10(4) to 2 x 10(6) s(-1). The Lewis-acid-catalyzed heterolysis reactions are fast enough for kinetic competence in coenzyme B12 dependent enzyme-catalyzed reactions of glycols, and Lewis-acid-catalyzed cleavages of beta-ethers in radicals might be applied in synthetic reactions.     

The elusive 5'-deoxyadenosyl radical in coenzyme-B12-mediated reactions

Vitamin B(12) and its biologically active counterparts possess the only examples of carbon-cobalt bonds in living systems. The role of such motifs as radical reservoirs has potential application in future catalytic and electronic nanodevices. To fully understand radical generation in coenzyme B(12) (dAdoCbl)-dependent enzymes, however, major obstacles still need to be overcome. In this work, we have used Car-Parrinello molecular dynamics (CPMD) simulations, in a mixed quantum mechanics/molecular mechanics (QM/MM) framework, to investigate the initial stages of the methylmalonyl-CoA-mutase-catalyzed reaction. We demonstrate that the 5'-deoxyadenosyl radical (dAdo(?)) exists as a distinct entity in this reaction, consistent with the results of extensive experimental and some previous theoretical studies. We report free energy calculations and first-principles trajectories that help understand how B(12) enzymes catalyze coenzyme activation and control highly reactive radical intermediates.     

喜树碱的电还原特性及应用研究

The voltammetric behavior of camptothecin （CPT） in Britton-Robinson （B-R） buffer solutions （pH 2.09-9.07） was studied by the means of linear sweep voltammetry （LSV）, cyclic voltarnmetry （CV） and normal pulse voltammetry （NPV） at a hanging mercury drop electrode. In different pH range of B-R buffer solutions, CPT could cause three reduction waves. In B-R buffer solutions （pH 2.09-5.46）, wave P1 yielded by CPT was a two-electron wave. Between pH 6.01 and 9.07, CPT could yield two reduction waves P2 and P3. In addition, the pure CPT obtained from camptotheca acumina grown only in China was determined by NPV, and a linear response was observed in the range of 2.0 × 10^-3-4.0 × 10^-2 mmol·L^-1 with a 0.9991 correlation coefficient and a 8.0 × 1^-4 mmol·L^-1 detection limit for CPT.     

分子模拟计算研究辅酶B12及其类似物光解产生的自由基加合物与β-环糊精的作用

辅酶Ｂ１２,即５′－脱氧腺苷钴铵素（ＡｄｏＢ１２）,作为辅因子参加生物体内多种酶反应的关键步骤是在酶诱导下ＡｄｏＢ１２的Ｃｏ－Ｃ键断裂产生５′－脱氧腺苷自由基ＡｄｏＣＨ２·。与许多Ｂ１２有关的酶反应相似,辅酶Ｂ１２及其类似物的光解反应也产生Ｂ１２ｒ和...     

Spectrophotometric determination of a nanomolar amount of ascorbic acid using its catalytic effect on copper(II) porphyrin formation

A simple, fast and sensitive flow-injection method is proposed for the determination of nanomolar amounts of ascorbic acid in tea, urine and blood. The procedure is based on the accelerating effect of a nanomolar level of ascorbic acid on the reaction of cooper(II) with 5,10,15,20-tetrakis(1-methylpyridinium-4-yl)porphyrin, H(2)tmpyp(4+). Ascorbic acid reduces Cu(II) to Cu(I) which catalyzes the incorporation of Cu(II) into H(2)tmpyp(4+) to form Cu(II)(tmpyp)(4+). In this method two solutions, one containing ascorbic acid and H(2)tmpyp(4+) and the other containing copper(II) and acetate buffer (pH 5.0), were injected into two flowing streams of water through two sample injectors of 120 mu1 sample volume. The mixture was allowed to react in a 2 m reaction coil and the colored solution of Cu(II)(tmpyp)(4+) was monitored at 550 nm (epsilon = 2.01 x 10(4)M(-1)cm(-1)). The present method was applied to the determination of ascorbic acid in tea, urea and blood. Reducing agents such as sugars and vitamins B(1), B(2), B(6) and B(12) did not give serious errors at a concentration of 10(-6) M for the determination of 1.0 x 10(-8)M ascrobic acid. The relative standard deviation of the present method was 2.8% for the determination of 1.0 x 10(-8)M ascorbic acid. The reaction mechanism was clarified from the kinetic results of the formation of Cu(II)(tmpyp)(4+) in the presence of various concentrations of ascorbic acid, copper(II) and hydrogen ion.     

Studies of CoQ10 and cyclodextrin complexes: solubility, thermo- and photo-stability

The complexes of coenzyme Q10 with β- and γ-cyclodextrin in aqueous solutions were prepared in order to improve the water solubility, thermo- and photo-stability of coenzyme Q10. Complex formation resulted in an increase in water solubility at room temperature and pH 6.5 by a factor at least 10². The solubility of coenzyme Q10 in the presence of cyclodextrins linearly increases with temperature and pH. The UV light (λ = 254) and temperature together have a great effect on coenzyme Q10 stability. After 120 min of exposure at 80 °C and UV light about 72.3% of pure coenzyme Q10 was degraded. Thermo- and photo-stability was strongly improved by complex formation; more than 64% of coenzyme Q10 remained unchanged. The formation of complexes was evaluated using IR spectrometry, X-ray diffractometry and TGA/DSC analysis.     

N-centered hexazirconium chloride clusters: excision and redox chemistry

The tightly cross-linked solid Zr(6)Cl(15)N yields [(Zr(6)NCl(12))Cl(6)](3-) upon heating with bis(triphenylphosphine)iminium chloride (PPNCl), in MeCN at 90 degrees C. Purple solutions containing [(Zr(6)NCl(12))Cl(6)](3-) were obtained and characterized with (15)N NMR. Cyclic voltammetric (CV) measurements on the series of [(Zr(6)ZCl(12))Cl(6)](n-) cluster ions (Z = Be, B, C, and N) in acetonitrile reveal that these cluster ions exhibit multiple reversible redox waves at potentials that can be systematically understood, including a reversible redox wave corresponding to the [(Zr(6)NCl(12))Cl(6)](3-/4-) couple. Preparation of the reduced cluster ion, [(Zr(6)NCl(12))Cl(6)](4-), (with 15 cluster-bonding electrons) was achieved by reduction of [(Zr(6)NCl(12))Cl(6)](3-) with (C(5)(CH(3))(5))(2)Co. Several new N-centered cluster complexes: (PPN)(3)[(Zr(6)NCl(12))Cl(6)].CH(2)Cl(2), [(C(5)(CH(3))(5))(2)Co(+)](3)[(Zr(6)NCl(12))Cl(6)], and (Et(4)N)(4)[(Zr(6)NCl(12))Cl(6)].2CH(3)CN have been isolated and structurally characterized.     

Mirror "base-off" conformation of coenzyme B12 in human adenosyltransferase and its downstream target, methylmalonyl-CoA mutase

Human adenosyltransferase synthesizes coenzyme B12, for the target mitochondrial B12 enzyme, methylmalonyl-CoA mutase. It binds B12 in the "base-off" conformation in both the Co2+ and Co3+ oxidation states as revealed by UV-visible and EPR spectroscopy although it lacks the signature DXHXXG motif found in other B12 proteins that bind the cofactor in this conformation. The "base-off" conformation, which is rare at physiological pH, mirrors that in the target enzyme, methylmalonyl-CoA mutase, which utilizes the product, AdoCbl. However, the coordination environment for cobalt in the two proteins is distinct, which is reflected in an approximately 40-fold difference in their affinity for the cofactor.     

Vitamin B12-derivatives-enzyme cofactors and ligands of proteins and nucleic acids

B(12)-cofactors play important roles in the metabolism of microorganisms, animals and humans. Microorganisms are the only natural sources of B(12)-derivatives, and the latter are "vitamins" for other B(12)-requiring organisms. Some B(12)-dependent enzymes catalyze complex isomerisation reactions, such as methylmalonyl-CoA mutase. They need coenzyme B(12), an organometallic B(12)-derivative, to induce enzymatic radical reactions. Another group of widely relevant enzymes catalyzes the transfer of methyl groups, such as methionine synthase, which uses methylcobalamin as cofactor. This tutorial review covers structure and reactivity of B(12)-derivatives and structural aspects of their interactions with proteins and nucleotides, which are crucial for the efficient catalysis by the important B(12)-dependent enzymes, and for achieving and regulating uptake and transport of B(12)-derivatives.     

Purification of the coenzyme B12-containing 2-methyleneglutarate mutase from Clostridium barkeri by high-performance liquid chromatography.

Two methods are described by which the enzymes 2-methyleneglutarate mutase and 3-methylitaconate delta-isomerase from Clostridium barkeri have been separated by high-performance liquid chromatography on a much larger scale than reported previously. First, the mutase eluted before the delta-isomerase after incubation with the mild detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonate (CHAPS) followed by high-performance anion-exchange chromatography on Mono Q in the presence of the same detergent. Second, an even better separation, although with a lower yield of mutase, was obtained by hydrophobic interaction chromatography on phenyl-Sepharose HiLoad, whereby the enzymes were eluted in the reverse order. Final high-performance anion-exchange chromatography of the latter preparation on Mono Q at pH 8 gave highly purified 2-methyleneglutarate mutase (greater than 95% purity) which had a pink-orange colour (lambda max 280, 375, 470 and 532 nm). The enzyme was active in the absence of coenzyme B12 (adenosylcobalamin) and contained 2.1 mol of this coenzyme per homotetramer (molecular mass, m = 300 kilodalton).