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 (alpha(6)) structure with one zinc atom per subunit. In the present work M(2+) binding and the role of Zn(2+) in EaCoMT have been established through a combination of biochemical, calorimetric, and spectroscopic techniques. A variety of metal ions, including Zn(2+), Co(2+), Cd(2+), and Ni(2+), were capable of activating a Zn-deficient "apo" form of EaCoMT, affording enzymes with various levels of activity. Titration of Co(2+) into apo-EaCoMT resulted in UV-visible spectroscopic changes consistent with the formation of a tetrahedral Co(2+) binding site, with coordination of bound Co(2+) 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 Co(2+) and Zn(2+) binding to EaCoMT also showed cooperativity for metal ion binding among six sites. The addition of CoM to Co(2+)-substituted EaCoMT resulted in UV-visible spectral changes indicative of formation of a new thiol-Co(2+) bond. Co(2+)-substituted EaCoMT exhibited a unique Co(2+) 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 Zn(2+), Co(2+), and Cd(2+) 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.
Boyd
Jeffrey M
JM
Department of Chemistry and Biochemistry, Utah State University, Logan, Utah 84322, USA.
Ensign
Scott A
SA
D001426:Bacterial Proteins; D001665:Binding Sites; D013437:Carbon-Sulfur Lyases / Q000378:metabolism*; D004852:Epoxy Compounds / Q000378:metabolism; D007700:Kinetics; D015080:Mesna / Q000378:metabolism*; D019216:Metals, Heavy / Q000378:metabolism*; D011407:Propane / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000378:metabolism
D001426:Bacterial Proteins; D004852:Epoxy Compounds; D019216:Metals, Heavy; D013438:Sulfhydryl Compounds; D013437:Carbon-Sulfur Lyases; C454421:2-hydroxypropyl-CoM 2-mercaptoethanesulfonate lyase (epoxyalkane-ring forming); D015080:Mesna; D011407:Propane