3-Methylaspartic acid as a potent antimetabolite of aspartic acid in pyrimidine biosynthesis.

The discovery last year that 3-methylaspartic acid (P-methylaspartic acid) is a natural metabolite formed by isomerization of glutamic acid through the intervention of a vitamin-Blz-containing coenzyme (1) suggests a new relationship of this vitamin to thymine biosynthesis. Thus, carbamylation of 3-methylaspartic acid would yield 3-methylureidosuccinic acid, which, by ring closure and deoxyribonucleotidation would yield dihydrothymidylic acid, and eventually thymidylic acid. These reactions would be quite analogous to those known to yield uridylic acid from aspartic acid, and would provide a new pathway for formation of thymidylic acid. We were particularly interested in this idea because we wished to make antimetabolites of 3-methylaspartic acid which might accordingly be expected to suppress thymine (or thymidylic acid) biosynthesis. We had shown earlier a relationship of vitamin B12 to certain spontaneous cancers (2, 3) and had been able to suppress some of them with antimetabolites which interfered with vitamin B12 formation (4, 5). An antimetabolite of 3-methylaspartic acid might be expected to enhance the efficiency of such compounds by providing a sequential blockade in reactions involving this vitamin and leading to deoxynucleotide synthesis. When Barker’s paper (1) appeared, attempts were therefore begun to synthesize and to test antimetabolites of a-methylaspartic acid. In the biological testing of some of these, the rather surprising finding was made that 3-methylaspartic acid itself was a very powerful inhibitor of the growth of Escherichia coli. Further study showed that this amino acid owed its toxic action on this bacterium to the fact that it was an antimetabolite of aspartic acid, and one which seemed to interfere with the functioning of aspartic acid in the formation of pyrimidine precursors. While the present manuscript was being prepared, a preliminary communication by Isenberg et al. (6) appeared which also set forth independently the idea that 3-methylaspartic acid might be a precursor of thymine. The evidence quoted was that this amino acid was able to replace the nutritional need for thymine and for vitamin Bit in certain microorganisms. In our work we had tested the ability of 3-methylaspartic acid to replace thymine for a thymine-requiring mutant of E. co& and had found it to be inactive. It would therefore seem that the block in biosynthesis in the mutant we used was at a different locus from that in the organisms of Isenberg et al. EXPERIMENTAL PROCEDURE