Enzymatic Synthesis of the Methyl Group of Methionine* IV. ISOLATION, CHARACTERIZATION, AND ROLE OF 5-METHYL TETRAHYDROFOLATE

The mechanism of formation de novo of the methyl group of methionine has been studied in mammalian, avian, and bacterial cell-free systems (l-8). The studies on extracts of various mutant strains of Escherichia coli have shown that, of the common compounds, formaldehyde or the P-carbon atom of serine is the best precursor of the methyl of methionine (5-7). Optimal formation of methionine from serine and homocysteine by extracts of mutant 113-3 of E. coli grown on a medium supplimented with vitamin Blz requires the participation of pyridoxal phosphate, a reduced folate derivative, reduced pyridine dinucleotide, ATP, magnesium ions, FAD, and at least three enzymes (5-7). Other enzyme components, however, have been implicated in the reaction (9-11). The first enzyme, serine hydroxymethylase, catalyzes the conversion of serine and folate-H4 to glycine and 5, IO-methylene folate-H4; pyridoxal phosphate is a cofactor for this reaction (7). Biosynthetic or chemically prepared 5, lo-methylene folate-Hr may substitute for the serine hydroxymethylase system (7). Two enzyme fractions, partially purified from E. coli extracts, convert 5, IO-methylene folate-H to methionine in the presence of homocysteine and the aforementioned cofactors. One of these required enzymes has a derivative of vitamin Blz as a prosthetic group, “B,, enzyme,” (7, 12) and another is absent in E. coli strain 205-2, “205-2 enzyme.” The preparation and properties of these two enzyme fractions have been the subject of the first two papers of this series (7, 13). This paper reports more detailed information on the order of action of these two enzymes and on the isolation and characterization of an intermediate of the over-all reaction, i.e. 5-methyl tetrahydrofolate. 5-Methyl tetrahydrofolate is synthesized from 5,lO methylene tetrahydrofolate and DPNH in the presence of the 205-2 enzyme, which now may be referred to as 5, lomethylene tetrahydrofolate reductase (Reaction 1) (14, 15). In the second step (Reaction 2) transmethylation occurs from 5methyl folate-& to homocysteine to yield methionine and folate-Hd. This reaction, as studied in the bacterial system, requires the participation of DPNH, FAD, ATP, Mg++, B,, enzyme (14, 16), and other protein fractions (9-11).