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

Electrostatic stress in catalysis: Structure and mechanism of the enzyme orotidine monophosphate decarboxylase

“…The remarkable catalytic power of ODCase, which accelerates the reaction by 17 orders of magnitude over the aqueous process, has fascinated chemists and biochemists alike, leading to a number of proposals of mechanisms with novel features (3)(4)(5)(6)(7). However, as more results accumulated for this class of enzymes, possibilities for the mechanism became increasingly limited as cofactors and catalytic groups continued to be excluded from consideration (8)(9)(10). The high-resolution x-ray structure of ODCase from Methanobacterium thermoautotrophicum reveals that the mechanism is almost fully characterized by the formal description, along with electrostatic features of the enzyme's active site that provide selective destabilization of the orotidine group.…”

A Reexamination of the Substrate Utilization of 2-Thioorotidine-5′-monophosphate by Yeast Orotidine-5′-Monophosphate Decarboxylase

“…We thus subjected our product to further analysis. 13 C NMR analysis of 2-thioorotate showed peaks with the following chemical shifts and their tentative assignments: 181.7 ppm (C2), 176.2 ppm (C7), 174.9 ppm (C4), 161.1 ppm (C6), 104.0 ppm (C5). The pattern is similar to, but distinct from, the 13 C signals from orotate, and no discernable traces of orotate signals in this preparation were evident.…”

“…NMR analysis of 2-thioorotate and 2-thioOMP. Synthesized 2-thioorotate was analyzed by 13 C NMR (proton decoupled), and 2-thioOMP analyzed by 31 P NMR, using a Varian Gemini 400 MHz NMR spectrometer. Samples of both compounds were prepared in D 2 O, with NaOD added to the 2-thioorotate to increase solubility.…”

“…In light of the new mechanism proposed, and to extend our previous finding that 2-thioUMP has essentially the same inhibition constant as UMP (13), a reexamination of the substrate utilization of 2-thioOMP seemed warranted. Possibly, the enzymatic synthesis method used previously (12) yielded a compound other than the anticipated product 2-thioOMP.…”

“…The striking result from thio-substitution studies is that OMP binding to the ODCase active site is essentially eliminated by 2-thio substitution, whereas UMP binding to the same active site is hardly affected by 2-thio substitution (13). In the four published crystal structures (1-4), there appears to be little steric constraint at O2 of each bound ligand, including O2 of UMP complexed to the Bacillus subtilis enzyme (1).…”