Indiscriminate Binding by Orotidine 5‘-Phosphate Decarboxylase of Uridine 5‘-Phosphate Derivatives with Bulky Anionic C₆ Substituents

Abstract

Orotidine 5'-phosphate (OMP) decarboxylase appears to act upon its substrate without the intervention of metals or other cofactors and without the formation of covalent bonds between the enzyme and the substrate. Crystallographic information indicates that substrate binding forces the substrate's scissile carboxylate group into the neighborhood of several charged groups at the active site. It has been proposed that binding might result in electrostatic stress at the substrate's C6 carboxylate group in such a way as to promote decarboxylation by destabilizing the enzyme-substrate complex in its ground state. If that were the case, one would expect uridine 5'-phosphate (UMP) derivatives with bulky anionic substituents at C6 to be bound weakly compared with UMP, which is unsubstituted at C6. Here, we describe the formation of anionic 5,6-dihydro-6-sulfonyl derivatives by spontaneous addition of sulfite to UMP and to OMP. These sulfite addition reactions, which are slowly reversible and are not catalyzed by the enzyme, result in the appearance of one (or, in the case of OMP, two) bulky anionic substituents at the 6-carbon atom of UMP. These inhibitors are bound with affinities that surpass the binding affinity of UMP. We are led to infer that the active site of OMP decarboxylase is remarkably accommodating in the neighborhood of C6. These are not the properties that one would expect of an active site with a rigid structure that imposes sufficient electrostatic stress on the substrate to produce a major advancement along the reaction coordinate.

Mechanism of the Orotidine 5′-Monophosphate Decarboxylase-Catalyzed Reaction: Evidence for Substrate Destabilization,

“…This structure also provides a possible explanation for Wolfenden's observation that derivatives of OMP and UMP obtained by addition of sulfite to the 5,6-double bond of the pyrimidine bases are effective inhibitors of ScOMPDC, with the values of their K i s lower than that for UMP (Scheme 4) (23). …”

Synthesis of rare nucleobases and artificial nucleotides for investigation of catalytic enzyme activity

“…However, it remains unclear if this effect is due to lower protonation at C2 and C4 position or worse fixation of the pyrimidine ring as a result of weaker hydrogen bonding to the thiocarbonyl groups. [44], [48], [49] A similar mechanism was proposed by Lee and Houk. They reported a protonation at O4 which gets stabilized as a carbene at C6, following decarboxylation.…”

Atomic Resolution Structure of the Orotidine 5′-Monophosphate Decarboxylase Product Complex Combined with Surface Plasmon Resonance Analysis

“…For 6-cyano-UMP, such a distortion receives strong support from Raman spectroscopy, which indicates bond bending of about 20°(15). The crystal structure of ODCase from Plasmodium falciparum in complex with OMP (16) The binding affinity of UMP, however, is significantly weaker than that of the substrate OMP and other UMP derivatives with negatively charged substituents at C6 (17)(18)(19)(20), an obviously serious argument against such an interpretation (18, 21). The low affinity of UMP seems inconsistent with the substrate distortion mechanism because UMP has lost its carboxylate group and with it the main candidate for repulsion with Asp-70.…”