A potential alternate substrate for orotidine-5Ј-monophosphate decarboxylase, 2-thio-orotidine-5Ј-monophosphate, was synthesized enzymatically and purified by a modification of a previous account (K. Shostak, and M. E. Jones 1992, Biochemistry 31, 12155-12161). Characterization of the product was confirmed by mass spectrometry, 31 P 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 Ϫ7 from the k cat for ODCase toward OMP. Additionally, 2-thioOMP effects no inhibition of ODCase decarboxylation of OMP at a concentration of 50 M, indicating a poor ability to bind to the ODCase active site. The results bear implications for proposed mechanisms for catalysis by ODCase. ᭧ 2001 Academic Press Recent protein crystal structures (1-4) have provided new insights into the catalytic mechanism of OMP decarboxylase (ODCase), 2 the enzyme with the highest measured catalytic proficiency (5). Originally, the catalytic mechanism of ODCase was presumed to proceed through a protonation step at O2 preceding decarboxylation (Fig. 1), yielding a zwitterionic intermediate, based on the observation of rates of uncatalyzed decarboxylation of model compounds (6). Support for this mechanism arose from enzyme inhibition (7) and isotope effect studies (8-10). A lysine residue (Lys93 of the yeast enzyme), common to all protein sequences of ODCases from many organisms, was found to be critical for catalysis and postulated as the general acid group