Activation of orotidine 5′-monophosphate decarboxylase by phosphite dianion: The whole substrate is the sum of two parts

We report that the binding of phosphite dianion to orotidine 5′-monophosphate decarboxylase (OMPDC) results in an 80000-fold increase in k_cat/K_m for decarboxylation of the truncated substrate, 1-(β-D-erythrofuranosyl)orotic acid (EO), which lacks a 5′-phosphodianion moiety. The intrinsic binding energy (IBE) of phosphite dianion in the transition state is 7.8 kcal/mol, which represents a very large fraction of the 11.8 kcal/mol IBE of the phosphodianion group of the natural substrate orotidine 5′-monophosphate (OMP). The data give k_cat = 160 ± 70 s^-1 for turnover of EO in the active site of OMPDC containing phosphite dianion, which is significantly larger than k_cat = 15 s^-1 for turnover of OMP. Despite the weaker binding of the individual EO and HPO₃^2- "parts" (K_mK_d = 0.014 M²) than of OMP (K_m = 1.6 × 10^-6 M), once bound, OMPDC provides a slightly greater stabilization of the transition state for reaction of the parts than of the whole substrate. Thus, the covalent connection between the reacting portion of the substrate and the nonreacting phosphodianion group is not necessary for efficient catalysis. This implies that a major role of the phosphodianion group of OMP is to provide binding interactions that are used to drive an enzyme conformational change, resulting in formation of an active site environment optimized for transition state stabilization.