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-(/sup 17/O, /sup 18/O)phosphoethanolamine ((/sup 17/O, /sup 18/O)DPPE) were synthesized and converted to (R/sub P/)- and (S/sub P/)-1,2-dipalmitoyl-sn-glycero-3-(/sup 16/O, /sup 17/O, /sup 18/O)DPPA), respectively, by incubating with phospholipase D. Condensation of (/sup 16/O, /sup 17/O, /sup 18/O)DPPA with cytidine 5'-monophosphomorpholidate in pyridine gave the desired substrate for PS synthase, (/sup 17/O, /sup 18/O)cytidine 5'-diphospho-1,2-dipalmitoyl-sn-glycerol ((/sup 17/O,/sup 18/O)CDP-DPG), as a mixture of several isotopic and configurational isomers. Incubation of (/sup 17/O, /sup 18/O)CDP-DPG), as a mixture of several isotopic and configurational isomers. Incubation of (/sup 17/O, /sup 18/O) CDP-DPG with a mixture of L-serine, PS synthase and PS decarboxylase gave (/sup 17/O, /sup 18/O)DPPE. The configuration and isotopic enrichments of the starting (/sup 17/O, /sup 18/O)DPPE and the product were analyzed by /sup 31/P 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.
- Research Organization:
- Univ. of Wisconsin, Madison
- OSTI ID:
- 5962927
- Journal Information:
- Biochemistry; (United States), Vol. 26:13
- Country of Publication:
- United States
- Language:
- English
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PHOSPHORUS-GROUP TRANSFERASES
CONFIGURATION INTERACTION
NUCLEAR MAGNETIC RESONANCE
STEREOCHEMISTRY
SUBSTRATES
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CHEMICAL SHIFT
ESCHERICHIA COLI
NMR SPECTRA
OXYGEN 16
OXYGEN 17
OXYGEN 18
PHOSPHOLIPIDS
PHOSPHORUS 31
PROTONS
SACCHAROMYCES CEREVISIAE
THIN-LAYER CHROMATOGRAPHY
BACTERIA
BARYONS
CARBON ISOTOPES
CHROMATOGRAPHY
ELEMENTARY PARTICLES
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EVEN-ODD NUCLEI
FERMIONS
FUNGI
HADRONS
ISOTOPES
LIGHT NUCLEI
LIPIDS
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NUCLEONS
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
ORGANIC PHOSPHORUS COMPOUNDS
OXYGEN ISOTOPES
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