Kinetic and microcalorimetric analysis of substrate and cofactor interactions in epoxyalkane:CoM transferase, a zinc-dependent epoxidase

Biochemistry. 2002 Apr 16;41(15):5005-14. doi: 10.1021/bi0255221.


Epoxyalkane:CoM transferase (EaCoMT) is a key enzyme of bacterial propylene metabolism, catalyzing the nucleophilic attack of coenzyme M (CoM, 2-mercaptoethanesulfonic acid) on epoxypropane to form the thioether conjugate 2-hydroxypropyl-CoM. The biochemical and molecular properties of EaCoMT suggest that the enzyme belongs to the family of alkyltransferase enzymes for which Zn plays a key role in activating an organic thiol substrate for nucleophilic attack on an alkyl-donating substrate. In the present work, the role of Zn in the EaCoMT-catalyzed reactions is established by removing Zn from EaCoMT, resulting in loss of catalytic activity that was restored upon addition of Zn back to the enzyme, and by expressing an inactive and Zn-deficient form of the enzyme that was activated by addition of ZnCl(2) or CoCl(2). Site-directed mutagenesis of one of the predicted Zn ligands (C220A) resulted in the formation of a largely catalytically inactive protein (0.06% of wild-type activity) that, when purified, contained a substoichiometric complement of Zn. EaCoMT was kinetically characterized and found to follow a random sequential mechanism with kinetic parameters K(m,epoxypropane) = 1.8 microM, K(m,CoM) = 34 microM, and k(cat) = 6.5 s(-1). The CoM analogues 2-mercaptopropionate, 2-mercaptoethanol, and cysteine substituted poorly for CoM as the thiol substrate, with specific rates of epoxyalkane conjugation that were at best 0.6% of the CoM-dependent rate, while ethanethiol, propanethiol, glutathione, homocysteine, and lipoic acid provided no activity. 2-Mercaptoethanol was a weak competitive inhibitor vs CoM with a K(I) of 192 mM. Isothermal titration calorimetry was used to investigate the thermodynamic binding determinants for the interaction of CoM and analogues with holo, Zn-deficient, and C220A EaCoMT variants. The stoichiometry of CoM binding correlated directly with the Zn content rather than monomer content of protein samples, reinforcing the importance of Zn in CoM binding. The binding of CoM to EaCoMT occurred with DeltaG = -7.5 kcal/mol (K(d) = 3.8 microM) and was driven by a large release of enthalpy. The thermodynamic contributors (K(a), DeltaG, DeltaH, DeltaS) to the individual binding of CoM, ethanesulfonate, and ethanethiol were determined and used to assess the contributions of the thiol, alkyl, and sulfonate moieties to total binding energy in the E x CoM binary complex.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Alkylation
  • Amino Acid Substitution
  • Calorimetry / methods
  • Carbon-Sulfur Lyases / chemistry
  • Carbon-Sulfur Lyases / metabolism*
  • Cations, Divalent / pharmacology
  • Chlorides / pharmacology
  • DNA Primers
  • Epoxide Hydrolases / chemistry
  • Epoxide Hydrolases / metabolism*
  • Hydrogen-Ion Concentration
  • Kinetics
  • Mercaptoethanol / pharmacology
  • Mesna / metabolism*
  • Mutagenesis, Site-Directed
  • Protein Binding
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Substrate Specificity
  • Xanthobacter / enzymology*
  • Zinc / pharmacology*
  • Zinc Compounds / pharmacology


  • Cations, Divalent
  • Chlorides
  • DNA Primers
  • Recombinant Proteins
  • Zinc Compounds
  • Mercaptoethanol
  • zinc chloride
  • Epoxide Hydrolases
  • Carbon-Sulfur Lyases
  • 2-hydroxypropyl-CoM 2-mercaptoethanesulfonate lyase (epoxyalkane-ring forming)
  • Zinc
  • Mesna