Thermolysis of coenzymes B 12 at physiological temperatures: activation parameters for cobalt–carbon bond homolysis and a quantitative analysis of the perturbation of the homolysis equilibrium by the ribonucleoside triphosphate reductase from Lactobacillus leichmannii

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The kinetics of the thermolysis of 5′-deoxyadenosylcobalamin (AdoCbl, coenzyme B 12) in aqueous solution, pH 7.5, have been studied in the temperature range 30–85°C using AdoCbl tritiated at the adenine C2 position and the method of initial rates. Combined with a careful analysis of the distribution of adenine-containing products, the results permit the dissection of the competing rate constants for carbon–cobalt bond homolysis and heterolysis. After correction for the temperature-dependent occurrence of the much less reactive base-off species of AdoCbl, the activation parameters for homolysis of the base-on species were found to be Δ H ‡ homo,on=33.8±0.2 kcal mol −1 and Δ S ‡ homo,on=13.5±0.7 cal mol −1 K −1, values not significantly different from those determined by Hay and Finke (J. Am. Chem. Soc. 108 (1986) 4820), in the temperature range 85–115°C. In contrast, the heterolysis of base-on AdoCbl was characterized by a much smaller enthalpy of activation (Δ H ‡ het,on=18.5±0.2 kcal mol −1) and a negative entropy of activation (Δ S ‡ het,on=−34.0±0.7 cal mol −1 K −1) so that heterolysis, which is minor pathway at elevated temperatures, is the dominant pathway for AdoCbl decomposition at physiological temperatures. Using literature values for the rate constant for the reverse reaction, the equilibrium constant for AdoCbl homolysis at 37°C was calculated to be 7.9×10 −18. Comparison with the equilibrium constant for this homolysis at the active site of the ribonucleoside triphosphate reductase from Lactobacillus leichmannii shows that the enzyme shifts the equilibrium constant towards homolysis products by a factor of 2.9×10 12 (17.7 kcal mol −1) by binding the thermolysis products with an equilibrium constant of 7.1×10 16 M −2, compared to the bonding constant for AdoCbl of 2.4×10 4 M −1.