A comparative study was made on the physicochemical characteristics of two isozymes of methylcobamide:-coenzyme M methyltransferase (MT2). Both isozymes catalyzed S-methylation of 2-thioethanesulfonate (coenzyme M) and exhibited similar apparent K(m) values for coenzyme M of 35 μM (MT2-A) and 20 μM (MT2-M). Weak binding to methylcobalamin was indicated by the apparent K(m) of 14 mM for both isozymes. Cob(I)alamin was established as the major product of the reaction, demonstrating heterolytic cleavage of the methylcobamide carbon-cobalt bond. The isozymes were shown to be zinc- containing metalloproteins. Metal ion chelators strongly inhibited both isozymes. A variety of coenzyme M analogs were tested for activity and/or inhibition. One alternative substrate 3-mercaptopropionate was discovered, with apparent K(m) 9 mM (MT2-A) and 10 mM (MT2-M). The results suggested an active site geometry in which coenzyme M is bound both by S-coordination to zinc, and electrostatic interaction of the sulfonate with a cationic group on the enzyme. Methanosarcina barkeri genes cmtA and cmtM encoding both isozymes were cloned and sequenced. Both genes encoded proteins with 339 amino acids and predicted molecular masses of 36-37 kDa. Active forms of both isozymes were expressed in Escherichia coli. A conserved segment with the potential for metal binding was found. The possibility of zinc involvement in catalysis of coenzyme M methylation is considered.
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