Kinetic and thermodynamic studies on ligand substitution reactions and base-on/base-off equilibria of cyanoimidazolylcobamide, a vitamin B12 analog with an imidazole axial nucleoside.
ABSTRACT Ligand substitution reactions of the vitamin B12 analog cyanoimidazolylcobamide, CN(Im)Cbl, with cyanide were studied. Cyanide substitutes imidazole (Im) in the alpha-position more slowly than it substitutes dimethylbenzimidazole in cyanocobalamin (vitamin B12). The kinetics of the displacement of Im by CN- showed saturation behaviour at high cyanide concentration; the limiting rate constant was found to be 0.0264 s(-1) at 25 degrees C and is characterized by the activation parameters: DeltaH(not =) = 111 +/- 2 kJ mol(-1), DeltaS(not =) = +97 +/- 6 J K(-1) mol(-1), and DeltaV(not =) = +9.3 +/- 0.3 cm3 mol(-1). These parameters are interpreted in terms of an I(d) mechanism. The equilibrium constant for the reaction of CN(Im)Cbl with CN- was found to be 861 +/- 75 M(-1), which is significantly less than that obtained for the reaction of cyanocobalamin with CN- (viz. 10(4) M(-1)). pKbase-off for the base-on/base-off equilibrium was determined spectrophotometrically and found to be 0.99 +/- 0.05, which is about 0.9 pH units higher than that obtained previously in the case of cyanocobalamin. In addition, the kinetics of the base-on/base-off reaction was studied using a pH-jump technique and the data obtained revealed evidence for an acid catalyzed reaction path. The results obtained in this study are discussed in reference to those reported previously for cyanocobalamin.
- SourceAvailable from: Rudi van EldikAdvances in Physical Organic Chemistry - ADVAN PHYS ORGAN CHEM. 01/2006; 41:1-78.
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ABSTRACT: Phenolyl cobamides are unique members of a class of cobalt-containing cofactors that includes vitamin B(12) (cobalamin). Cobamide cofactors facilitate diverse reactions in prokaryotes and eukaryotes. Phenolyl cobamides are structurally and chemically distinct from the more commonly used benzimidazolyl cobamides such as cobalamin, as the lower axial ligand is a phenolic group rather than a benzimidazole. The functional significance of this difference is not well understood. Here we show that in the bacterium Sporomusa ovata, the only organism known to synthesize phenolyl cobamides, several cobamide-dependent acetogenic metabolisms have a requirement or preference for phenolyl cobamides. The addition of benzimidazoles to S. ovata cultures results in a decrease in growth rate when grown on methanol, 3,4-dimethoxybenzoate, H(2) + CO(2), or betaine. Suppression of native p-cresolyl cobamide synthesis and production of benzimidazolyl cobamides occurs upon the addition of benzimidazoles, indicating that benzimidazolyl cobamides are not functionally equivalent to the phenolyl cobamide cofactors produced by S. ovata. We further show that S. ovata is capable of incorporating other phenolic compounds into cobamides that function in methanol metabolism. These results demonstrate that S. ovata can incorporate a wide range of compounds as cobamide lower ligands, despite its preference for phenolyl cobamides in the metabolism of certain energy substrates. To our knowledge, S. ovata is unique among cobamide-dependent organisms in its preferential utilization of phenolyl cobamides.Journal of bacteriology 02/2013; · 2.69 Impact Factor
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ABSTRACT: Cobamides, which include vitamin B12 (cobalamin), are a class of modified tetrapyrroles synthesized exclusively by prokaryotes that function as cofactors for diverse biological processes. Cobamides contain a centrally bound cobalt ion that coordinates to upper and lower axial ligands. The lower ligand is covalently linked to a phosphoribosyl moiety through an alpha-glycosidic bond formed by the CobT enzyme. CobT can catalyze the phosphoribosylation of a variety of substrates. We investigated the ability of CobT to act on either of two nitrogen atoms within a single, asymmetric benzimidazole substrate to form two isomeric riboside phosphate products. Reactions containing asymmetric benzimidazoles as substrates for homologs of CobT from different bacteria resulted in the production of distinct ratios of two isomeric products, with some CobT homologs favoring the production of a single isomer and others forming a mixture of products. These preferences were reflected in the production of cobamide isomers with lower ligands attached in different orientations, some of which are novel cobamides that have not been characterized previously. Two isomers of methoxybenzimidazolylcobamide were found to be unequal in their ability to support ethanolamine ammonia-lyase dependent growth in Salmonella enterica, suggesting that CobT's regiospecificity could be biologically important. We also observed differences in pKa, which can influence the reactivity of the cofactor and could contribute to these distinct biological activities. Relaxed regiospecificity was achieved by introducing a single point mutation in an active site residue of CobT. These new cobamide isomers could be used to probe the mechanisms of cobamide-dependent enzymes.Biochemistry 11/2014; · 3.38 Impact Factor