Zinc-thiolate intermediate in catalysis of methyl group transfer in Methanosarcina barkeri.
Methyl group transfer reactions are essential in methane-forming pathways in all methanogens. The involvement of zinc in catalysis of methyl group transfer was studied for the methyltransferase enzyme MT2-A important for methanogenesis in Methanosarcina barkeri growing on methylamines. Zinc was shown to be required for MT2-A activity and was tightly bound by the enzyme with an apparent stability constant of 10(13.7) at pH 7.2. Oxidation was a factor influencing activity and metal stoichiometry of purified MT2-A preparations. Methods were developed to produce inactive apo MT2-A and to restore full activity with stoichiometric reincorporation of Zn(2+). Reconstitution with Co(2+) yielded an enzyme with 16-fold higher specific activity. Cysteine thiolate coordination in Co(2+)-MT2-A was indicated by high absorptivity in the 300-400 nm charge transfer region, consistent with more than one thiolate ligand at the metal center. Approximate tetrahedral geometry was indicated by strong d-d transition absorbance centered at 622 nm. EXAFS analyses of Zn(2+)-MT2-A revealed 2S + 2N/O coordination with evidence for involvement of histidine. Interaction with the substrate CoM (2-mercaptoethanesulfonic acid) resulted in replacement of the second N/O group with S, indicating direct coordination of the CoM thiolate. UV-visible spectroscopy of Co(2+)-MT2-A in the presence of CoM also showed formation of an additional metal-thiolate bond. Binding of CoM over the range of pH 6.2-7.7 obeyed a model in which metal-thiolate formation occurs separately from H(+) release from the enzyme-substrate complex. Proton release to the solvent takes place from a group with apparent pK(a) of 6.4, and no evidence for metal-thiolate protonation was found. It was determined that substrate metal-thiolate bond formation occurs with a Delta G degrees ' of -6.7 kcal/mol and is a major thermodynamic driving force in the overall process of methyl group transfer.
Gencic
S
S
Department of Biochemistry and Molecular Biology, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, Maryland 20814-4799, USA.
LeClerc
G M
GM
Gorlatova
N
N
Peariso
K
K
Penner-Hahn
J E
JE
Grahame
D A
DA
D002384:Catalysis; D003035:Cobalt / Q000737:chemistry / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D015080:Mesna / Q000737:chemistry / Q000378:metabolism; D008670:Metals / Q000378:metabolism; D017021:Methanosarcina barkeri / Q000737:chemistry*; D008780:Methyltransferases / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D013053:Spectrophotometry; D013438:Sulfhydryl Compounds / Q000737:chemistry*; D013816:Thermodynamics; D013997:Time Factors; D014965:X-Rays; D015032:Zinc / Q000737:chemistry* / Q000378:metabolism
D008670:Metals; D013438:Sulfhydryl Compounds; D003035:Cobalt; D006861:Hydrogen Peroxide; D008780:Methyltransferases; C098666:methylcobamide-coenzyme M methyltransferase; D015032:Zinc; D015080:Mesna