Quantum catalysis in B ₁₂ -dependent methylmalonyl-CoA mutase: experimental and computational insights

Abstract

B 12 -dependent methylmalonyl-CoA mutase catalyses the interchange of a hydrogen atom and the carbonyl-CoA group on adjacent carbons of methylmalonyl-CoA to give the rearranged product, succinyl-CoA. The first step in this reaction involves the transient generation of cofactor radicals by homolytic rupture of the cobalt-carbon bond to generate the deoxyadenosyl radical and cob(II)alamin. This step exhibits a curious sensitivity to isotopic substitution in the substrate, methylmalonyl-CoA, which has been interpreted as evidence for kinetic coupling. The magnitude of the isotopic discrimination is large and a deuterium isotope effect ranging from 35.6 at 20 8C to 49.9 at 5 8C has been recorded. Arrhenius analysis of the temperature dependence of this isotope effect provides evidence for quantum tunnelling in this hydrogen transfer step. The mechanistic complexity of the observed rate constant for cobalt-carbon bond homolysis together with the spectroscopically silent nature of many of the component steps limits the insights that can be derived by experimental approaches alone. Computational studies using a newly developed geometry optimization scheme that allows determination of the transition state in the full quantum mechanical/molecular mechanical coordinate space have yielded novel insights into the strategy deployed for labilizing the cobalt-carbon bond and poising the resulting deoxyadenosyl radical for subsequent hydrogen atom abstraction.

Coupling of hydrogenic tunneling to active-site motion in the hydrogen radical transfer catalyzed by a coenzyme B12-dependent mutase

“…The most probable reason for the differing conclusions seems to be the extreme sensitivity of the system to the size of the model and the details of the dynamical model (5,16,21). Our experience indicates that only inclusion of a substantial part of the enzyme environment yields results that are pertinent to the enzymecatalyzed reaction (22).…”

Synthesis, solution and crystal structure of the coenzyme B12 analogue Coβ-2′-fluoro-2′,5′-dideoxyadenosylcobalamin

“…While H-bonds were clearly seen as crucial, the present crystal structure analyses still failed to explain how the Co-C-bond homolysis was achieved at the rate observed for the enzymatic processes, and how the intermediate radicals were stabilized (see e.g. [24]). However, the crystal structure of GLM indicated a conformational reorientation of the Ado moiety from C2′-endo to C3′-endo, elongating the Co-C-bond from 3.2 Å to about 4.5 Å, and thus activating it further for an effective homolysis.…”

Integrating computational methods to retrofit enzymes to synthetic pathways

“…3c). Following previously reported QM and QM/MM protocols for MCM (Banerjee, 2003; Banerjee et al, 2006; Jensen and Ryde, 2005; Kwiecien et al, 2006; Loferer et al, 2003; Rovira and Biarne, 2004; Sharma et al, 2007), we compute the free energy profile for the homolysis reaction, using TI methods (see the Supplementary Results for more information). The barrier height of the reaction is computed to be 12.45 kcal mol −1 .…”