Orotidine-5-Monophosphate Decarboxylase: Purification and spectral studies, /

Abstract

The chemical mechanism by which orotidylate decarboxylase (ODCase) catalyzes the formation of uridylate (UMP) from orotidylate (OMP) is apparently different from any of the common decarboxylation routes seen with other enzymes. ODCase is the final enzyme of the de novo pyrimidine biosynthetic pathway and it displays one of the largest rate enhancements ever measured. Different mechanisms have been suggested to explain the mechanistic action of ODCase. Our group suggested a model for this decarboxylation that involves proton donation to the transition state in a concerted protonationldecarboxylation mechanism and possible participation of a Zn+z ion. Yeast ODCase was purified from any contaminating proteins whereas its mutant (K93C) was partially purified. Thio-substituted analogues were used as inhibitors to probe the active site of the enzyme. The inhibition constants of these compounds were measured by 14COZ displacement assays, and their pKa's were determined by spectrophotometric assays. IH NMR spectroscopy of ODCase in complex with barbituric acid ribonucleotide (BMP) at different DzO concentrations and UMP at 10% DzO reveals downfield signals, possibly identifying hydrogen bonds between the enzyme and the inhibitor. The two farthest downfield signals in the spectra are absent in the free ODCase spectrum. Our proposed catalytic mechanism and the results obtained from the experiments are to be discussed in this thesis.