The second order rate constant (k(cat)/K(m)) for decarboxylation of orotidine by yeast OMP decarboxylase (ODCase), measured by trapping (14)CO(2) released during the reaction, is 2 x 10(-4)M(-1)s(-1). This very low activity may be compared with a value of 3 x 10(7)M(-1)s(-1) for the action of yeast OMP decarboxylase on the normal substrate OMP. Both activities are strongly inhibited by 6-hydroxy UMP (BMP), and abrogated by mutation of Asp-96 to alanine. These results, in conjunction with the binding affinity of inorganic phosphate as a competitive inhibitor (K(i)=7 x 10(-4)M), imply an effective concentration of 1.1 x 10(9)M for the substrate phosphoryl group in stabilizing the transition state for enzymatic decarboxylation of OMP. The observed difference in rate (1.5 x 10(11)-fold) is the largest effect of a simple substituent that appears to have been reported for an enzyme reaction.