A kinetic study of the immediate nucleotide precursor of riboflavin in whole cells of Eremothecium ashbyii at rest.

Acid -soluble nucleotides from the mycelia were clearly separated by column chromatography on Dowex 1 X 2(HCOO-, 200 to 400 mesh). Under the experimental conditions used [2--14C] xanthine and [2--14C] guanine added were not incorporated into adenosine nucleotides but only into the guanosine nucleotides-GMP, GDP, GDP-Man, and GpA. Purines labeled at carbon 2 were effectively transferred to riboflavin but not radioactivity from [8--14C]--hypoxanthine was detected in the produced riboflavin. Comparison of the specific activity/time curves of guanosine nucleotides and riboflavin indicated that the specific activity of newly formed riboflavin coincides perfectly with that of GTP during incubation, and that the specific activity of accumulated riboflavin, at its maximum value, intersects the GTP curve. Thus, the kinetic studies with whole cells of E. ashbyii provide clear evidence that, among various nucleotides, GTP is the immediate precursor of riboflavin, and further attest that the intermediates involved in the biosynthetic pathway of the nucleotide precursor to riboflavin are in a trace amounts but have high turnover rates and, that the biosynthetic pathway has no salvage pathway for the intermediate derivatives, di- and tri-amino pyrimidines.