Is There a Dynamic Protein Contribution to the Substrate Trigger in Coenzyme B12-Dependent Ethanolamine Ammonia Lyase?
Is There a Dynamic Protein Contribution to the Substrate Trigger in Coenzyme B12-Dependent Ethanolamine Ammonia Lyase?
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DOI：
10.1002/anie.201105132
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发表时间：
2011-01-01
影响因子：
16.6
通讯作者：
Scrutton, Nigel S.
中科院分区：
文献类型：
--
作者：
Jones, Alex R.;Hardman, Samantha J. O.;Scrutton, Nigel S.
Coenzyme B12, or 5о-deoxyadenosylcobalamin (AdoCbl), acts as cofactor to a number of enzymes from a range of organisms.[1, 2] In all cases, the CoÀC bond in the cofactor undergoes homolysis upon substrate binding, generating a singlet-born, CblII/adenosyl radical pair (RP) and thus initiating radical-mediated catalysis. When compared to thermal homolysis of the free cofactor in solution,[3] rate increases achieved by these enzymes are in the region of 1011–1013,[4–6] the precise origin of which is not yet fully understood. To date, the protein contribution to this catalytic power has been discussed either in terms of ground-state destabilization and a “strain” hypothesis,[7] or transition state stabilization by electrostatic factors.[8] However, there may be another contribution to consider—protein dynamics. Using a unique combination of spin-chemical and photochemical techniques we present evidence for coupling between RP reaction dynamics and protein dynamics in AdoCbl-dependent ethanolamine ammonia lyase (EAL). The adenosyl radical has never been observed directly during turnover in an AdoCbl-dependent enzyme under ambient conditions. In EAL it is rapidly quenched by H-abstraction from the substrate to give the more stable substrate radical.[6, 9, 10] The CoÀC bond can be photolyzed,[11] however, enabling investigation of the singlet-born geminate pair dynamics at room temperature both in the free and protein-bound cofactor. The spin-state of this RP can coherently interconvert between the singlet and the triplet sublevels (Scheme 1). If the geminate RP re-encounter, only those in the singlet state will recombine, whereas triplet pairs will separate again.[12] The extent to which the spin-states mix can be altered by the application of external magnetic fields (MFs).[13, 14] For increasing MFs of moderate strength (tens to hundreds of mT) the TÆ1 levels are gradually removed in energy and ultimately only S and T0 interconvert. With a singlet-born RP, this process increases the relative S population and hence the probability of recombination. Such magnetic field effects (MFEs) have been observed in the rate of anaerobic, continuous wave (cw) photolysis of both free and EAL-bound AdoCbl.[15, 16] Under continuous illumination the reactive adenosyl radicals are ultimately and irreversibly quenched to yield an accumulated CblII signal, and the MFE manifests as a decrease in the apparent rate of this accumulation. The magnitude of the MFE was viscositydependent for unbound AdoCbl, the viscogen acting as a RP “cage”. Likewise, the protein limits RP diffusion, thus enhancing the MFE over that observed in buffered water.[16] The magnetic sensitivity of homolysis is removed in EAL, however, when the CoÀC bond is broken thermally by substrate binding.[6] The observation of a significant kinetic isotope effect in the pre-steady-state signal representing the conversion of AdoCblIII to CblII suggests kinetic coupling of homolysis to subsequent H-abstraction from the substrate. The effect of this coupling is to rapidly quench the adenosyl radical, generating the substrate radical (which accumulates during turnover),[10] thus stabilizing against recombination of the geminate pair and removing the MFE. However, this does not preclude the possibility of MF-sensitivity in the recombination step after product release.[17] While the chemistry that immediately follows homolysis in the EAL-catalyzed reaction appears to favor RP dissociation, what of the protein contribution? The role of protein dynamics in enzyme function [18, 19] is commonly probed by varying solvent viscosity (see, eg Ref.[20, 21]) and assessing the extent to which this variation at …
