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|Title:||Component C of the Methylcoenzyme M Methylreductase|
|Author(s):||Hartzell, Patricia Louise|
|Department / Program:||Microbiology|
|Degree Granting Institution:||University of Illinois at Urbana-Champaign|
|Abstract:||Component C, an enzyme in the methylcoenzyme M methylreductase complex of methanogens, has been proposed to contain the catalytic site for methyl group reduction from CH(,3)-S-CH(,2)CH(,2)SO(,3)('-) (methylcoenzyme M). Component C, a hexamer in an (alpha)(,2), (beta)(,2), (gamma)(,2) configuration, contains a chromophore F(,430), coenzyme M and component B (7-mercaptoheptanoylthreonine phosphate). There are two mol of F(,430), a unique nickel tetrapyrrole, per mol of component C. Coenzyme M, the C-1 carrier in the terminal step of methanogenesis, is also present in a ratio of two mol per mol of protein.
The role of the nickel tetrapyrrole, F(,430), in catalysis was tested by isolating and purifying F(,430) and each of the subunits of component C. Recovery of maximal enzyme activity required each subunit, F(,430), and coenzyme M. When F(,430) was omitted from the reassociation mixture, the M(,r) 300,000 apoprotein was formed, but was unable to function in the methanogenic assay. Isomers of the nickel tetrapyrrole differed in their ability to reassociate with the enzyme subunits and yielded holoenzyme with altered specific activities. Salt extracted F(,430), a six-coordinate form, gave optimal reassociation and specific activity whereas the four-coordinate heat extracted F(,430) reassociated poorly with the enzyme subunits.
The function of the bound C-1 carrier, coenzyme M, in the enzyme was examined with radiolabel tracing during in vitro methanogenesis using a double labeled substrate, C('3)H(,3)-S-CH(,2)CH(,2)('35)SO(,3)('-). The bound form of the coenzyme was found to be HS-CH(,2)CH(,2)SO(,3)('-) and the stoichiometry of 2 mol of HS-CoM per mol of enzyme was confirmed. The incorporation was slow and was not stoichiometric with methane production during catalysis. The incorporation was specific for the thioethanesulfonate moiety of CH(,3)-S-CoM; labeled HS-CoM was not incorporated into the enzyme during methanogenesis.
The substrate analogue, bromoethanesulfonate (BES), was found to competitively inhibit methanogenesis from methylcoenzyme M. The target of inhibition was component C.
Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1987.
|Date Available in IDEALS:||2014-12-16|