Stereochemical course of phosphokinases. The use of adenosine [.gamma.-(S)-16O,17O,18O]triphosphate and the mechanistic consequences for the reactions catalyzed by glycerol kinase, hexokinase, pyruvate kinase, and acetate kinase

Abstract

The synthesis of adenosine [.gamma.-(S)-16O,17O,18O]triphosphate, an isotopically labeled species of ATP that is chiral at the .gamma.-phosphoryl group, the configuration of which was confirmed by independent stereochemical analysis is reported. This molecule was used as a substrate in the reactions catalyzed by glycerol kinase and acetate kinase [from Escherichia coli]. The resulting samples of isotopically labeled sn-glycerol 3-phosphate and acetyl phosphate were used as substrates in the alkaline phosphatase mediated transfer of the chiral phosphoryl groups to (S)-propane-1,2-diol, from which the configuration at P was determined. Glycerol kinase and acetate kinase (and by virtue of an earlier correlation [muscle] pyruvate kinase and [yeast] hexokinase) proceed by pathways that result in inversion of the configuration at P. The stereochemical approach provides an access to the otherwise cryptic events that are involved in phosphoryl-group transfer within the ternary complexes of these kinases and their substrates.