Atomic details of near-transition state conformers for enzyme phosphoryl transfer revealed by ... formula ... rather than by phosphoranes

摘要

Prior evidence supporting the direct observation of phosphorane intermediates in enzymatic phosphoryl transfer reactions was based on the interpretation of electron density corresponding to trigonal species bridging the donor and acceptor atoms. Close examination of the crystalline state of β-phosphoglucomutase, the archetypal phosphorane intermediate-containing enzyme, reveals that the trigonal species is not PO3-, but is MgF3- (trifluoromagnesate). Although MgF3- complexes are transition state analogues rather than phosphoryl group transfer reaction intermediates, the presence of fluorine nuclei in near-transition state conformations offers new opportunities to explore the nature of the interactions, in particular the independent measures of local electrostatic and hydrogen-bonding distributions using F19 NMR. Measurements on three β-PGM-MgF3--sugar phosphate complexes show a remarkable relationship between NMR chemical shifts, primary isotope shifts, NOEs, cross hydrogen bond F ⋯ H-N scalar couplings, and the atomic positions determined from the high-resolution crystal structure of the β-PGM-MgF3--G6P complex. The measurements provide independent validation of the structural and isoelectronic MgF3- model of near-transition state conformations.