X-ray Crystallographic Studies of the R65Q Mutant of Yeast Phosphoglycerate Kinase

Author: McPhillips, Timothy M.

Year: 1997

Degree: Dissertation (Ph.D.)

Advisor: Rees, Douglas C.

Committee Members: Beauchamp, Jesse L.; Rees, Douglas C.; Barton, Jacqueline K.; Goddard, William A., III

Option: Chemistry

DOI: 10.7907/p1pg-0748

Abstract

The structure of a ternary complex of the R65Q mutant of yeast 3-phosphoglycerate kinase (PGK) with magnesium 5'-adenylylimido-diphosphate (Mg-AMP-PNP) and 3- phospho-D-glycerate (3-PG) was determined by X-ray crystallography to 2.4 Å resolution. The structure was solved by single isomorphous replacement, anomalous scattering and solvent flattening, and has been refined to an R-factor of 0.186, with rms deviations from ideal bond distance and angles of 0.009 Å and 1.82°, respectively. The structure represents the first view of a ternary complex of PGK with both substrates. The 3-PG is located in a 'basic patch' of residues on the N-terminal domain, while the Mg-AMP-PNP interacts with residues on the C-terminal domain. The overall structure is an 'open' configuration with the two substrates separated by ~ 11 Å across the interdomain cleft. The structure is inconsistent with the hinge-bending model of PGK function which predicts a closed configuration for the ternary complex. Based on the available structural, biochemical, and site-directed mutagenesis data, it is proposed that the basic patch primarily represents the site of anion activation and not the catalytically active binding site for 3-PG. The active site of PGK is proposed to be the combination of the nucleotide binding site on the C-domain and a possible 3-PG binding site between the domains, near the N-terminus of helix 14 and the sidechain of arginine-38. The kinetic properties and crystal structure of the R65Q mutant of yeast PGK are consistent with this proposal.

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