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 t o one of the carbonyl oxygens of the substrate, and may also include partic ipation of a Zn2+ ion. To probe the active site for these mechanistic featu res, thio-substituted analogues of the product UMP were used as inhibitors of yeast ODCase. The intrinsic inhibition constants of the anionic pyrimidi nes were calculated using the measured inhibition constants and the pK(a) v alues of the respective compounds. 4-ThioUMP is a stronger inhibitor than U MP, while 2-thioUMP has a K-i virtually the same as that for UMP. A potenti al alternate substrate, CMP-6-carboxylate, has been synthesized and found t o have undetectable activity and weak binding to ODCase. The results are di scussed in a unified model for catalysis involving protonation at O2 and a proposed Zn2+ interaction at O4, (C) 1999 Academic Press.