The role of a global, substrate-driven, enzyme conformational change in enabling the extraordinarily large rate acceleration for orotidine 5’-monophosphate decarboxylase (OMPDC)-catalyzed decarboxylation of orotidine 5’-monophosphate (OMP) is examined in experiments that focus on the interactions between OMPDC and the ribosyl hydroxyl groups of OMP. The D37 and T100’ side chains of OMPDC interact, respectively, with the C-3’ and C-2’ hydroxyl groups of enzyme-bound OMP. D37G and T100’A substitutions result in 1.4 kcal/mol increases in the activation barrier ΔG(‡) for catalysis of decarboxylation of the phosphodianion truncated substrate 1-(β-D-erythrofuranosyl)orotic acid (EO), but in larger 2.1–2.9 kcal/mole increases in ΔG(‡) for decarboxylation of OMP, and for phosphite dianion-activated decarboxylation of EO. This shows that these substitutions reduce transition state stabilization by the Q215, Y217 and R235 side chain at the dianion binding site. The D37G and T100’A substitutions result in
