Pyrimidine biosynthesis in Serratia marcescens: polypeptide interactions of three nonsequential enzymes

Orotidine-5'-monophosphate pyrophosphorylase (OMPppase, E.C. 2.4.2.10) and orotidylate decarboxylase (OMPdecase, E.C. 4.1.1.23) were purified from Serratia marcescens HY. These enzymes required physical association for maximal catalytic activities and formed a fragile complex with dihydroorotase (DHOase, E.C. 3.5.2.3). OMPppase reversibly lost 50% of its activity upon separation from DHOase. The kinetic characteristics of OMPppase were modified by this separation. In the presence of DHOase, the Kms for PRPP and orotate were stoichiometric: 2.3 X 10(-6) M and 2.6 X 10(-6) M, respectively. Following separation, the Kms were significantly different: 1.3 X 10(-6) M for PRPP and 4.1 X 10(-6) M for orotate. OMPppase and OMPdecase could be reversibly separated by acrylamide gel electrophoresis, but the separation was accompanied by a loss of catalytic efficiency for both enzymes. DHOase readily associated into multiple molecular forms and could not be purified. The DHOase-OMPppase-OMPdecase interactions demonstrate that a weakly aggregated, multifunctional enzyme complex participates in the biosynthesis of pyrimidine nucleotides in S. marcescens. This unique association of non-sequential biosynthetic enzymes may represent a larger complex which provides a channeling or regulatory unit.