LIGAND GEOMETRY OF THE TERNARY COMPLEX OF 5-ENOLPYRUVYLSHIKIMATE-3-PHOSPHATE SYNTHASE FROM ROTATIONAL-ECHO DOUBLE-RESONANCE NMR

The 46-kDa enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase c atalyzes the condensation of shikimate 3-phosphate (S3P) and phosphoen olpyruvate (PEP) to form EPSP. The reaction is inhibited by N-(phospho nomethyl)glycine (Glp), which, in the presence of S3P, binds to EPSP s ynthase to form a stable ternary complex. As part of a solid-state NMR characterization of this structure, we have used dipolar recovery at the magic angle (DRAMA) and rotational-echo double resonance (REDOR) t o determine intra- and interligand internuclear distances. DRAMA was u sed to determine the single P-31-P-31 distance, while REDOR was used t o determine one P-31-N-15 distance and five P-31-C-13 distances. These experimental distances were used as restraints in molecular dynamics simulations of an S3P-Glp complex to examine the geometry of the two l igands relative to one another in the ternary complex. The simulations were compared to unrestrained simulations of the EPSP synthase tetrah edral intermediate and its phosphonate analog. The results suggest tha t Glp is unlikely to bind in the same fashion as PEP, a conclusion tha t is consistent with recent studies that have questioned the role of G lp as a transition-state or intermediate analog.