| Abstrakt: |
A potential alternate substrate for orotidine-5′-monophosphate decarboxylase, 2- thio-orotidine-5′-monophosphate, was synthesized enzymatically and purified by a modification of a previous account (K. Shostak, and M. E. Jones 1992, Biochemistry31, 12155–12161). Characterization of the product was confirmed by mass spectrometry, 31P NMR, and utilization by orotate phosphoribosyltransferase in the direction of pyrophosphorolysis. The previous work probably did not result in the purification of the desired compound, as evidenced by our observation of 2-thioOMP's sensitivity to high temperature, as used previously. Using a very sensitive HPLC assay for the potential decarboxylated product 2-thioUMP, no measurable activity of ODCase toward the alternate substrate was observed, representing a decarboxylation rate decreased by 10−7 from the kcat for ODCase toward OMP. Additionally, 2-thioOMP effects no inhibition of ODCase decarboxylation of OMP at a concentration of 50 μM, indicating a poor ability to bind to the ODCase active site. The results bear implications for proposed mechanisms for catalysis by ODCase. |
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