ASSIGNMENT OF ENZYMATIC FUNCTION TO SPECIFIC PROTEIN REGIONS OF COBALAMIN-DEPENDENT METHIONINE SYNTHASE FROM ESCHERICHIA-COLI

Cobalamin-dependent methionine synthase catalyzes methyl group transfe r from methyltetrahydrofolate to homocysteine to form tetrahydrofolate and methionine, and the cobalamin prosthetic group serves as an inter mediate methyl carrier. Enzyme possessing cobalamin in the cobalt(II) oxidation state is inactive, and this form is activated by one-electro n reduction coupled to methylation by S-adenosylmethionine (AdoMet). T he enzyme from Escherichia coli has been divided into separable fragme nts by limited proteolysis with trypsin, and the contribution of each of these fragments to substrate binding and catalysis has been evaluat ed. The 37.7-kDa carboxyl-terminal domain binds AdoMet, and this was d emonstrated through covalent modification with radiolabeled AdoMet dur ing ultraviolet irradiation. Following reductive activation with AdoMe t, the enzyme was digested with trypsin and a 98.4-kDa amino-terminal fragment was isolated. It retained at least 70% of the activity of the intact enzyme and must therefore possess determinants sufficient for the binding of methyltetrahydrofolate and homocysteine, as well as res idues required for catalysis. However, when the cobalamin was oxidized to the cob(II)alamin state, the 98.4-kDa fragment could not be reduct ively remethylated with AdoMet. A purified, 28-kDa domain within the 9 8.4-kDa fragment retained bound cobalamin and therefore must play a ce ntral role in catalysis, but the isolated 28-kDa domain retained no ca talytic activity. Because AdoMet binds to a different domain of the pr otein than methyltetrahydrofolate and homocysteine, the enzyme probabl y uses conformational flexibility to allow the cobalamin access to the required methyl donor or acceptor at the appropriate time in catalysi s. These results provide an explanation for the exclusion of AdoMet fr om the normal turnover cycle and suggest how AdoMet, methyltetrahydrof olate, and homocysteine may be bound simultaneously.