Orotidine 5′-phosphate decarboxylase (ODase) catalyzes the conversion of orotidylate to uridylate, the last step in the de novo biosynthesis of pyrimidine nucleotides. Model reactions are described that support a covalent catalytic mechanism for this enzyme in which, following protonation of the carboxyl group of orotidylic acid, an active-site nucleophile undergoes a Michael addition to the C-5 position. This covalent complex breaks down via an acid-base-catalyzed decarboxylative elimination reaction to give uridylate and CO2(Scheme II). The enzyme mechanism is modeled in two parts, the Michael addition reaction and the decarboxylative elimination. Bisulfite is shown to undergo a Michael addition to N,N-dimethylorotaldehyde and at room temperature to N,N-dimethyl-6-acetyluracil, both models for the activated form of orotidylate, the substrate for ODase (6 → 7). In a separate study, (±)-1,3-dimethyl-r-5-(methylthio)-5-methyl-trans-6-carboxyl-5,6-dihydrouracil (15) was prepared as a model for the ODase-orotidylate covalent complex. Activation by methylation of the sulfide (as a model for enzyme-catalyzed protonation) leads to instantaneous decarboxylative elimination at room temperature. When the corresponding ester (9c) is methylated, the dimethylsulfonium salt (16b) can be isolated, which upon ester hydrolysis gives the decarboxylative elimination product. These model studies support the Michael addition-decarboxylative elimination mechanism in favor of a noncovalent mechanism previously reported.
ASJC Scopus subject areas
- Colloid and Surface Chemistry