Stereochemistry of phosphoryl transfer

Area of Science:

• Biochemistry
• Organic Chemistry
• Enzymology

Background:

• Chiral phosphate esters are crucial in biological systems.
• Understanding the stereochemical course of enzymatic phosphoryl transfer is vital for mechanistic insights.

Purpose of the Study:

• To develop a general method for synthesizing chiral [16O,17O,18O]phosphate monoesters with known absolute configuration.
• To establish an analytical method for determining the absolute configuration of these chiral phosphate esters.
• To investigate the stereochemical outcome of phosphoryl transfer reactions catalyzed by specific enzymes.

Main Methods:

• Synthesis of chiral [16O,17O,18O]phosphate monoesters.
• Determination of absolute configuration using 17O and 18O isotope effects at phosphorus in 31P nuclear magnetic resonance (NMR) spectroscopy.
• Enzymatic assays using hexokinase, phosphofructokinase, and pyruvate kinase.

Main Results:

• Successful synthesis of chiral [16O,17O,18O]phosphate monoesters with defined absolute configurations.
• Establishment of a reliable NMR-based method for determining the absolute configuration of chiral phosphate esters.
• Demonstration that phosphoryl transfer catalyzed by hexokinase, phosphofructokinase, and pyruvate kinase proceeds with inversion of configuration.

Conclusions:

• The developed methods provide a powerful tool for studying the stereochemistry of phosphoryl transfer.
• Enzymatic phosphoryl transfer reactions catalyzed by the studied kinases occur with inversion of configuration.
• The results support an "in-line" phosphoryl group transfer mechanism within the enzyme-substrate ternary complex.