Epoxyalkane:coenzyme M transferase (EaCoMT) catalyzes the nucleophilic addition of coenzyme M (CoM, 2-mercaptoethanesulfonic acid) to epoxypropane forming 2-hydroxypropyl-CoM. The biochemical properties of EaCoMT suggest that the enzyme belongs to the family of alkyltransferase enzymes for which Zn plays a role in activating an organic thiol substrate for nucleophilic attack on an alkyl-donating substrate. The enzyme has a hexameric (α6) structure with one zinc atom per subunit. In the present work M2+ binding and the role of Zn2+ in EaCoMT have been established through a combination of biochemical, calorimetric, and spectroscopic techniques. A variety of metal ions, including Zn2+, Co2+, Cd 2+, and Ni2+, were capable of activating a Zn-deficient "apo" form of EaCoMT, affording enzymes with various levels of activity. Titration of Co2+ into apo-EaCoMT resulted in UV-visible spectroscopic changes consistent with the formation of a tetrahedral Co 2+ binding site, with coordination of bound Co2+ to two thiolate ligands. Quantification of UV-visible spectral changes upon Co 2+ titration into apo-EaCoMT demonstrated that EaCoMT binds Co 2+ cooperatively at six interacting sites. Isothermal titration calorimetric studies of Co2+ and Zn2+ binding to EaCoMT also showed cooperativity for metal ion binding among six sites. The addition of CoM to Co2+-substituted EaCoMT resulted in UV-visible spectral changes indicative of formation of a new thiol-Co2+ bond. Co 2+-substituted EaCoMT exhibited a unique Co2+ EPR spectrum, and this spectrum was perturbed significantly upon addition of CoM. The presence of a divalent metal ion was required for the release of protons from CoM upon binding to EaCoMT, with Zn2+, Co2+, and Cd2+ each facilitating proton release. The divalent metal ion of EaCoMT is proposed to play a key role in the coordination and deprotonation of CoM, possibly through formation of a metal-thiolate that is activated for attack on the epoxide substrate.
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