Purification and Properties of Orotidine-5′-Phosphate Pyrophosphorylase and Orotidine-5′-Phosphate Decarboxylase from Bakers' Yeast

Orotidine-5′-phosphate (O5P) pyrophosphorylase [EC 2. 4. 2.10] and O5P decarboxylase [EC 4.1.1. 23] were separately purified from bakers' yeast about 1, 800- and 6, 000-fold, respectively. O5P pyrophosphorylase activity was resolved into major and minor components on DEAE-cellulose. Molecular weights were estimated to be 39, 000 for the main- and 32, 000 for the minor-component of O5P pyrophosphorylase, and 51, 000 for O5P decarboxylase from their elution positions on Sephadex G-100 gel filtration. Purified O5P pyrophosphorylase had the pH optimum at 8.5, generally stable at pH above 7.5, and catalyzed both the O5P formation (forward reaction) and the O5P pyrophosphorolysis (reverse reaction) at about the same initial rate. The Km values were estimated to be 33 μM for orotate, 62μM for 5-phosphoribosylpyrophosphate, 8.3μM for O5P and 220 μM for PP1. Its enzymatic activity or its stability was not affected by the presence of sulfhydryl compounds or sulfhydryl reagents. On the other hand, purified O5P decarboxylase had the pH optimum at 5.5, generally stable at pH below 7.5, and unstable in a frozen state. The enzyme was not fully active in the absence of sulfhydryl compounds, and fully activated by cysteine, reduced glutathione (GSH), dithiothreitol (DTT) or β-mercaptoethanol. For the long storage at 4°C, however, β-mercaptoethanol was most effective in protecting the enzyme from inactivation. This enzyme was also sensitive to sulhydryl reagents like p-chloromercuribenzoate and 5, 5’-dithiobis (2-nitrobenzoic acid). The forward reaction of O5P pyrophosphorylase was markedly inhibited by O5P, the product, but not inhibited by most of other nucleotides. In contrast, O5P decarboxylase was moderately inhibited by GMP, GDP and CMP, but not inhibited by other nucleotides including uridine nucleotides. The Km value for orotate was estimated to be 5μM, and the K1 values were calculated to be 1.2 mM for GMP and 0.28 mM for CMP.