Crystalline Pyruvate Oxidase from Escherichia coli: II. ACTIVATION BY PHOSPHOLIPIDS

Abstract

Abstract The activity of pyruvate oxidase from Escherichia coli increases 15- to 20-fold in the presence of phospholipids extracted from cell membranes. Fractionation and analysis of the phosphatides from E. coli cell envelope preparations revealed that lysophosphatidylethanolamine exhibited highest specific activity for stimulating the flavoprotein if the phospholipids were added directly to the assay mixtures. However, when water-soluble micellar preparations were substituted for direct addition of the phospholipid to the assay, all the phosphatides demonstrated higher specific activities for stimulating pyruvate oxidase. Furthermore, the differences originally noted in the activating capacities of the various cell envelope phospholipids were minimized. By measuring pyruvate oxidase activity under conditions in which the micellar phospholipid preparation was rate-limiting, Michaelis constants (Km) for various phosphatides were determined. The Km values for the cell envelope phospholipids, synthetic phosphatidylethanolamine, lecithin, and lysolecithin ranged from 0.9 to 2.2 x 10-6 m. The Km value for phosphatidylserine was somewhat higher, being 6.5 x 10-6 m. The diacylphospholipids exhibited normal Michaelis-Menten kinetics. Lysophosphatides demonstrated considerable divergence from normal Michaelis-Menten kinetics. This observation suggests that pyruvate oxidase differentiates between phospholipids according to the number of fatty acids contained within the molecular structure. The hydrophobic moieties of lecithin activate pyruvate oxidase whereas the hydrophilic portions of the molecule have no stimulatory effect. The enzyme can also be activated by fatty acids at concentrations only slightly higher than those required for maximal stimulation by micellar phospholipid preparations. Of the fatty acids tested, palmitoleic and oleic appear to be most efficient in stimulating pyruvate oxidase activity.