Active Site Probes for Yeast OMP Decarboxylase: Inhibition Constants of UMP and Thio-Substituted UMP Analogues and Greatly Reduced Activity toward CMP-6-Carboxylate

The catalytic mechanism of orotidine-5′-monophosphate decarboxylase (ODCase, EC 4.1.1.23) involves a proton-sensitive step, probably proton donation to one of the carbonyl oxygens of the substrate, and may also include participation of a Zn2+ ion. To probe the active site for these mechanistic features, thio-substituted analogues of the product UMP were used as inhibitors of yeast ODCase. The intrinsic inhibition constants of the anionic pyrimidines were calculated using the measured inhibition constants and the pKa values of the respective compounds. 4-ThioUMP is a stronger inhibitor than UMP, while 2-thioUMP has a Ki virtually the same as that for UMP. A potential alternate substrate, CMP-6-carboxylate, has been synthesized and found to have undetectable activity and weak binding to ODCase. The results are discussed in a unified model for catalysis involving protonation at O2 and a proposed Zn2+ interaction at O4.