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.     

Polarography of B(12) coenzyme

The polarography of B(12) coenzyme (5,6-dimethylbenzimidazolylcobamide, DBC coenzyme) has been investigated at the dropping mercury electrode. Exposure of solutions of the coenzyme to light and then to oxygen give polarograms comparable to those of B(12r) and hydroxocobalamin (B(12a)) respectively. At pH 11.6 the coenzyme gives two reduction waves, at -1.43 and -1.62 V vs. the S.C.E.; in less basic solutions the two waves merge to give one multielectron wave.     

Characterization of organic substrates bound to cross-linked polystyrenes by 13C NMR spectroscopy

The ¹³C NMR spectra of a wide variety of organic substrates bound to 2% cross-linked polystyrenes may be obtained routinely, provided the resins can be sufficiently swollen. The ¹³C chemical shifts of polymer-bound trityl alcohol, polymer-bound monotrityl ethers of the symmertrical diols HO (CH₂)_nOH (n=2, 4, 6, 7, 9 and 10), and some related intermediates in the solid phase synthesis of insect pheromones are presented. ¹³C shift additivity correlations, differing little from those in free trityl ethers, are drawn.     

Contribution of uniformly 13C-enriched sterigmatocystin to the study of its pulmonary metabolism

Mycotoxins are secondary metabolites of filamentous fungi which can cause a wide range of systemic effects. Human health effects of inhaled mycotoxins remain poorly documented, despite the large amounts present, associated with air-borne particles. Among these mycotoxins, sterigmatocystin is one of the most prevalent. Because its chemical structure is close to that of the aflatoxins, we studied its metabolism and its cellular consequences when in contact with the airway epithelium, using the mass spectral signature from the 10% (13)C uniformly enriched sterigmatocystin. The metabolism was studied in vitro, using recombinant cytochrome P450s enzymes, and in porcine tracheal epithelial cell (PTEC) primary cultures at an air-liquid interface. The metabolites were analyzed by high-performance liquid chromatography coupled with tandem mass spectrometry detection. Expressed enzymes and PTECs were exposed to uniformly (13)C-enriched sterigmatocystin to confirm the relationship between sterigmatocystin and its metabolites because this isotopic cluster shape is conserved for all metabolites and their product ions. Incubation of sterigmatocystin with recombinant cytochrome P450 1A1 led to the formation of three metabolites identified as monohydroxysterigmatocystin, dihydroxysterigmatocystin and one glutathione adduct, the latter after the formation of a transient intermediate. In the PTEC cultures, sterigmatocystin metabolism resulted in a glucuro-conjugate. Two other products were detected, a sulfo-conjugate and a glucuro-conjugate of hydroxysterigmatocystin upon cytochrome P450 1A1 induction. This is the first study to report sterigmatocystin metabolism in airway epithelium, and it suggests that, contrary to the aflatoxins, sterigmatocystin is mainly detoxified into its conjugates and is unable to produce significant amounts of reactive metabolites in respiratory cells, at least in pigs.     

Assignment of the NMR spectra of a potent ribonucleotide reductase inactivator - utilization of HMQC-TOCSY to establish connectivities in exchange broadened spectra

Inverted and Suppressed Direct Response (IDR and SDR, respectively) HMQC-TOCSY experiments are evaluated relative to the conventional HMQC-TOCSY experiment in the assignment of the congested proton and carbon spectra of a 2-acetylpyridine thiocarbonohydrazone used to potentiate the antiviral drug acyclovir. Spectra with moderate overlap can be most expeditiously interpreted using the inverted direct response HMQC-TOCSY variant; spectra with severe overlap can be more readily interpreted if direct responses are suppressed.     

Dynamic nuclear polarization-enhanced solid-state NMR of a 13C-labeled signal peptide bound to lipid-reconstituted Sec translocon

Dynamic nuclear polarization (DNP) has made it possible to record 2D double-quantum-filtered (DQF) solid-state NMR (ssNMR) spectra of a signal peptide bound to a lipid-reconstituted SecYEG translocon complex. The small quantity of peptide in the sample (~40 nmol) normally prohibits multidimensional ssNMR experiments. Such small amounts are not the exception, because for samples involving membrane proteins, most of the limited sample space is occupied by lipids. As a consequence, a conventional 2D DQF ssNMR spectrum with the sample used here would require many weeks if not months of measurement time. With the help of DNP, however, we were able to acquire such a 2D spectrum within 20 h. This development opens up new possibilities for membrane protein studies, particularly in the exploitation of high-resolution spectroscopy and the assignment of individual amino acid signals, in this case for a signal peptide bound to the translocon complex.     

Ligand substitution behavior of a simple model for coenzyme B12

The ligand substitution reactions of trans-[CoIII(en)2(Me)H2O]2+, a simple model for coenzyme B12, were studied for cyanide and imidazole as entering nucleophiles. It was found that these nucleophiles displace the coordinated water molecule trans to the methyl group and form the six-coordinate complex trans-[Co(en)2(Me)L]. The complex-formation constants for cyanide and imidazole were found to be (8.3 +/- 0.7) x 10(4) and 24.5 +/- 2.2 M-1 at 10 and 12 degrees C, respectively. The second-order rate constants for the substitution of water were found to be (3.3 +/- 0.1) x 10(3) and 198 +/- 13 M-1 s-1 at 25 degrees C for cyanide and imidazole, respectively. From temperature and pressure dependence studies, the activation parameters delta H++, delta S++, and delta V++ for the reaction of trans-[CoIII(en)2(Me)H2O]2+ with cyanide were found to be 50 +/- 4 kJ mol-1, 0 +/- 16 J K-1 mol-1, and +7.0 +/- 0.6 cm3 mol-1, respectively, compared to 53 +/- 2 kJ mol-1, -22 +/- 7 J K-1 mol-1, and +4.7 +/- 0.1 cm3 mol-1 for the reaction with imidazole. On the basis of reported activation volumes, these reactions follow a dissociative mechanism in which the entering nucleophile could be weakly bound in the transition state.     

The reduction of ribonucleotides catalyzed by the enzyme ribonucleotide reductase

This work is devoted to the study of the radical catalytic pathway for the ribonucleotide reduction process assisted by ribonucleotide reductase. The present study is directed toward the investigation of one of the most controversial steps in the reduction pathway – the elimination of the 2′ hydroxyl group from the ribonucleotide. Several groups have made different proposals for this step, which all fit available experimental data; however, so far, it has not been possible to demonstrate clearly which is the correct pathway for the elimination. Here, we resort to high-level quantum mechanical calculations to analyze the energetics of the proposed mechanisms, as well as to propose alternative pathways, and evaluate their feasibility, according to the observed kinetics of the enzyme and other existing experimental data. Our study shows that the elimination occurs via two different simultaneous acid-/base-catalyzed pathways, depending on the protonation state of one key active site amino acid, Glu441.     

New developments in the field of vitamin B12: enzymatic reactions dependent upon corrins and coenzyme B12.

Simple corrins such as vitamin B₁₂ and vitamin B₁₂ coenzyme catalyze a variety of unusual enzymatic reactions of which some are still without analogy in organic or organometallic chemistry. The mechanisms of these reactions are currently the subject of lively discussion. The present review focuses attention on new ideas about the mode of action of vitamin B₁₂ coenzymes in enzymatic reactions.     

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).     

Carboxylate-substituted radicals from phenylselenide derivatives. Designs on models for coenzyme B12-dependent enzyme-catalyzed rearrangements

Laser flash photolysis (266 nm) of alpha- and beta-phenylselenyl esters, carboxylic acids, and carboxylates in aqueous acetonitrile media gave the corresponding radicals by homolytic cleavage of the phenylselenyl groups. In the beta-substituted systems, acid and carboxylate radicals reacted in intramolecular reporter reactions with approximately equal rate constants. For the alpha-substituted systems, an ester- and carboxylic acid-substituted radical reacted in an intramolecular reporter reaction with the same rate constants, but the analogous alpha-carboxylate radical, a radical anion, reacted an order of magnitude less rapidly and with an activation energy that is 3 kcal/mol greater than that found for analogues. A kinetic titration of the equilibrating alpha-acid and alpha-carboxylate radicals gave pKa = 4.6. The results indicate that alpha-ester and alpha-carboxylic acid radicals are unlikely to be appropriate models for alpha-carboxylate radicals, the intermediates formed in a large subset of coenzyme B12-dependent enzyme-catalyzed reactions.     

1H NMR isotope shifts arising from the substitution of 12C by 13C: application to polycyclic aromatic hydrocarbon anions

Isotope shifts are a well-established tool for structural analysis by NMR. The substitution of a protium with a deuterium is the most widely studied of these effects. However, such studies call for specific deuteration that requires complex synthetic techniques owing to the low natural abundance of deuterium. ¹³C occurs at a higher natural abundance and couples strongly with its attached proton. We have developed and refined a method to reliably observe these much smaller shifts without needing to resort to chemical synthesis. We show that carbon induced isotope shifts reflect structural features. Copyright © 2001 John Wiley & Sons, Ltd.     

13C NMR spectroscopic studies on the conformation during stepwise synthesis of peptides bound to solubilizing polymer supports

The C-13 {C-13-H-1} triple resonance technique allows constitutional and conformational studies on polypeptides bound to mono- and bi-functional solubilizing polyoxyethylene supports. High resolution shows distinct differences between random coil and α-helical conformations. This method is a valuable additional tool for control of the growing peptide during stepwise synthesis on soluble polymeric supports. Examples of partial sequences of the polypeptide antibiotic alamethicin and sequential analogs (Aib-Ala)_n demonstrate the applicability for peptide polyoxyethylene esters with mean molecular masses between 2000 and 10,000.