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

Abstract

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, k obs, 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, K b, 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, K eq, for its formation from enzyme-bound AdoCbl, can be calculated. These values of K eq have been used to deconvolute the forward, k f, and reverse, k r, rate constants from the measured k obs 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 k f and van't Hoff plots of K eq 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 (ΔH f ⧧ = 20 ± 1 kcal mol-1, ΔS f ⧧ = 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.

Solution structure, enzymatic, and non-enzymatic reactivity of 3-isoadenosylcobalamin, a structural isomer of coenzyme B12 with surprising coenzymic activity

“…The results (Fig. 5) were qualitatively similar to those previously obtained for AdoCbl [47] and for two analogs with altered lower axial nucleotides [68]. As before, the observed first-order rate constants for the rapid, reversible formation of cob(II)alamin were independent of the enzyme concentration, but the absorbance change, DA, indicative of the extent of formation of cob(II)alamin, increased with RTPR concentration and appears to ''saturate''.…”

“…Steady-state RTPR assays utilized the coupled spectrophotometric method [46], with ATP as the substrate and dGTP as allosteric effector, as previously described [47]. Pre-steady-state kinetic measurements of the enzyme-induced formation of cob(II)alamin were made on an Applied Photophysics SX.17MV stopped flow spectrophotometer equipped with an AN1 anaerobic accessory.…”

“…Pre-steady-state kinetic measurements of the enzyme-induced formation of cob(II)alamin were made on an Applied Photophysics SX.17MV stopped flow spectrophotometer equipped with an AN1 anaerobic accessory. Measurements were made at 525 nm using DTT as reductant, under strictly anaerobic conditions and at 37°C as described elsewhere [47].…”

“…The kinetics of formation of cob(II)alamin upon mixing RTPR and 3-IsoAdoCbl were measured spectrophotometrically using DTT as the reductant, dGTP as allosteric effector, and no NTP substrate [66,67], as described previously [47,68]. The results (Fig.…”

“…The consensus structure has the 3-IsoAdo ligand over the eastern quadrant, but in the syn conformation. [47,68], the simplest interpretation of these results is that rapid coenzyme binding (K b ) is followed by the observable reversible cleavage of the carbon-cobalt bond (k f and k r , Eq. (2)) ð2Þ Eq.…”