Phospholipids chiral at phosphorus. Steric course of the reactions catalyzed by phosphatidylserine synthase from Escherichia coli and yeast

The steric courses of the reactions catalyzed by phosphatidylserine (PS) synthase from Escherichia coli and yeast were elucidated by the following procedure. R/sub P/ and S/sub P/ isomers of 1,2-dipalmitoyl-sn-glycero-3-[17O, 18O]phosphoethanolamine ([17O, 18O]DPPE) were synthesized and converted to (R/sub P/)- and (S/sub P/)-1,2-dipalmitoyl-sn-glycero-3-[16O, 17O, 18O]DPPA), respectively, by incubating with phospholipase D. Condensation of [16O, 17O, 18O]DPPA with cytidine 5'-monophosphomorpholidate in pyridine gave the desired substrate for PS synthase, [17O, 18O]cytidine 5'-diphospho-1,2-dipalmitoyl-sn-glycerol ([17O,18O]CDP-DPG), as a mixture of several isotopic and configurational isomers. Incubation of [17O, 18O]CDP-DPG), as a mixture of several isotopic and configurational isomers. Incubation of [17O, 18O] CDP-DPG with a mixture of L-serine, PS synthase and PS decarboxylase gave [17O, 18O]DPPE. The configuration and isotopic enrichments of the starting [17O, 18O]DPPE and the product were analyzed by 31P NMR following trimethylsilylation of the DPPE. The results indicate that the reaction of E. coli PS synthase proceeds with retention of configuration at phosphorus, which suggests a two-step mechanism involving a phosphatidyl-enzyme intermediate, while the yeast PS synthase catalyzes the reaction with inversion of configuration, which suggests a single-displacement mechanism. Such results lend strong support to the ping-pong mechanism proposed for the E. coli enzyme and the sequential Bi-Bi mechanism proposed for the yeast enzyme, both based on previous isotopic exchange experiments