Evidence for a metal-thiolate intermediate in alkyl group transfer from epoxypropane to coenzyme M and cooperative metal ion binding in epoxyalkane:CoM transferase

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.