Activation Parameters for the Carbon−Cobalt Bond Homolysis of Coenzyme B12Induced by the B12-Dependent Ribonucleotide Reductase fromLactobacillus leichmannii

The temperature dependence of the kinetics of the rapid, reversible cleavage of the carbon−cobalt bond of 5'-deoxyadenosylcobalamin (AdoCbl, coenzyme B12) catalyzed by the ribonucleotide triphosphate reductase (RTPR) of Lactobacillus leichmannii has been studied by stopped-flow spectrophotometry. At a given temperature and constant concentration of AdoCbl, the observed rate constants, kobs, are essentially independent of the initial concentration of RTPR, but the spectral change, and hence the amount of cob(II)alamin formed at equilibrium, shows a hyperbolic dependence on [RTPR]0. This is interpreted as being a consequence of the known relatively weak binding of AdoCbl to the enzyme so that increasing enzyme concentration drives the binding equilibrium toward enzyme−coenzyme complex. Fitting the absorbance change data to a binding isotherm gives values of the binding equilibrium constant, Kb, and the maximal absorbance change obtainable when the coenzyme is "saturated" with enzyme. From the latter value and the difference in molar absorptivity of AdoCbl and cob(II)alamin, the concentration of enzyme-bound cob(II)alamin, and consequently the equilibrium constant, Keq, for its formation from enzyme-bound AdoCbl, can be calculated. These values of Keq have been used to deconvolute the forward, kf, and reverse, kr, rate constants from the measured kobs values for cob(II)alamin formation at the active site. The enzyme is found to catalyze the Co−C bond homolysis of AdoCbl by a factor of 1.6 × 109 (ΔΔG⧧ = 13 kcal mol-1) at 37 °C. Eyring plots of kf and van't Hoff plots of Keq both show distinct discontinuities at about 32 °C which may indicate the formation of an inactive, or less active, conformer at lower temperature or a change in mechanism. The activation parameters for the forward rate constant for cob(II)alamin in the upper temperature regime (ΔHf⧧ = 20 ± 1 kcal mol-1, ΔSf⧧ = 13 ± 4 cal mol-1 K-1) show that the entropy of activation is essentially the same as that for the nonenzymatic thermal homolysis of AdoCbl, but the enthalpy of activation is 13 ± 2 kcal mol-1 lower. The enzyme thus appears to catalyze the Co−C bond cleavage reaction entirely enthalpically. Several possibilities for the enthalpic catalysis of AdoCbl homolysis by RTPR are discussed.