Two enzymic mechanisms for the methylation of homocysteine by extracts of Escherichia coli.

Earlier work in this Laboratory has established conditions for the formation of methionine, from homocysteine plus serine or formaldehyde, by soluble enzyme systems extracted from Eseherichia coli. A cofactor of the folic acid group* is essential, and with E. coli PA 15 an extract of the heated organisms, H4PtG3 and its N5and N'0-formyl derivatives are all effective. H4PtG inhibits competitively the action of both H4PtG3 and the extract of heated E. coli (Szulmajster & Woods, 1960; Jones, Guest & Woods, 1961); such inhibition does not, however, occur if cobalamin is added either to the ultrasonic extracts or to the medium on which the organisms are grown (Kisliuk & Woods, 1960; Guest, Helleiner, Cross & Woods, 1960). With ultrasonic extracts of E. coli 121/176 (an auxotroph requiring cobalamin or methionine for growth) cobalamin is essential even when H4PtG3 (or extract of heated E. coli) is present. Cobalamin acts after conversion into a complex factor (Kisliuk & Woods, 1960; Guest & Woods, 1960) which has been purified from strain PA15 and shown to be a cobamide-containing enzyme (Foster, Jones & Woods, 1961a; Kisliuk, 1961). Analogues of cobalamin inhibit the formation of the cobamide-containing enzyme from cobalamin or 5,6-dimethylbenzimidazolylcobamide coenzyme, but are without effect on the activity of extracts of organisms grown in the presence ofadded cobalamin (Guest, 1960) and presumably possessing the cobamide-containing enzyme. The enzymic synthesis of methionine from homocysteine and serine with H4PtG as cofactor has also been studied in E. coli 113/3 (another cobalamin or methionine auxotroph) and found to require a cobamide-containing enzyme which is formed from cobalamin and an apoenzyme present in extracts of autotrophic organisms grown in the absence of cobalamin (Hatch, Takeyama & Buchanan, 1959; Hatch, Cathou & Larrabee, 1960; Takeyama & Buchanan, 1960). Extracts of mammalian liver form methionine with H4PtG (or its N5-formyl derivative) as