Assignment of enzymatic function to specific protein regions of cobalamin-dependent methionine synthase from Escherichia coli.
Cobalamin-dependent methionine synthase catalyzes methyl group transfer from methyltetrahydrofolate to homocysteine to form tetrahydrofolate and methionine, and the cobalamin prosthetic group serves as an intermediate methyl carrier. Enzyme possessing cobalamin in the cobalt(II) oxidation state is inactive, and this form is activated by one-electron reduction coupled to methylation by S-adenosylmethionine (AdoMet). The enzyme from Escherichia coli has been divided into separable fragments by limited proteolysis with trypsin, and the contribution of each of these fragments to substrate binding and catalysis has been evaluated. The 37.7-kDa carboxyl-terminal domain binds AdoMet, and this was demonstrated through covalent modification with radiolabeled AdoMet during ultraviolet irradiation. Following reductive activation with AdoMet, 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 residues required for catalysis. However, when the cobalamin was oxidized to the cob(II) alamin state, the 98.4-kDa fragment could not be reductively remethylated with AdoMet. A purified, 28-kDa domain within the 98.4-kDa fragment retained bound cobalamin and therefore must play a central role in catalysis, but the isolated 28-kDa domain retained no catalytic activity. Because AdoMet binds to a different domain of the protein than methyltetrahydrofolate and homocysteine, the enzyme probably uses conformational flexibility to allow the cobalamin access to the required methyl donor or acceptor at the appropriate time in catalysis.(ABSTRACT TRUNCATED AT 250 WORDS)
Drummond
J T
JT
Department of Biological Chemistry, University of Michigan, Ann Arbor 48109.
Huang
S
S
Blumenthal
R M
RM
Matthews
R G
RG
D013766:5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology*; D006710:Homocysteine / Q000378:metabolism; D012436:S-Adenosylmethionine / Q000378:metabolism; D013763:Tetrahydrofolates / Q000378:metabolism; D014805:Vitamin B 12 / Q000378:metabolism*
D013763:Tetrahydrofolates; D006710:Homocysteine; D012436:S-Adenosylmethionine; D013766:5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase; D014805:Vitamin B 12; C005984:5-methyltetrahydrofolate