STRUCTURE OF THE R65Q MUTANT OF YEAST 3-PHOSPHOGLYCERATE KINASE COMPLEXED WITH MG-AMP-PNP AND 3-PHOSPHO-D-GLYCERATEFE

The structure of a ternary complex of the R65Q mutant of yeast 3-phosp hoglycerate kinase (PGK) with magnesium 5'-adenylylimidodiphosphate (M g AMP-PNP) and 3-phospho-D-glycerate (3-PG) has been determined by X-r ay crystallography to 2.4 Angstrom resolution, The structure was solve d by single isomorphous replacement, anomalous scattering, and solvent flattening and has been refined to an R-factor of 0.185, with rms dev iations from ideal bond distance and angles of 0.009 Angstrom and 1.78 degrees, respectively. PGK consists of two domains, with the 3-PG bou nd to a ''basic patch'' of residues from the N-terminal domain and the Mg-AMP-PNP interacting with residues from the C-terminal domain. The two ligands are separated by similar to 11 Angstrom across the interdo main cleft, The model of the R65Q mutant of yeast PGK is very similar to the structures of PGK isolated from horse, pig, and Bacillus stearo thermophilus (rms deviations between equivalent ex-carbons in the indi vidual domains < 1.0 Angstrom) but exhibits substantial variations wit h a previously reported yeast structure (rms deviations between equiva lent alpha-carbons in the individual domains of 2.9-3.2 Angstrom). The most significant tertiary structural differences among the yeast R65Q , equine, porcine, and B. stearothermophilus PGK structures occur in t he relative orientations of the two domains. However, the relationship s between the observed conformations of PGK are inconsistent with a '' hinge-bending'' behavior that would close the interdomain cleft. It is proposed that the available structural and biochemical data on PGK ma y indicate that the basic patch primarily represents the site of anion activation and not the catalytically active binding site for 3-PG.