Nuclear‐Magnetic‐Resonance Study of the Active‐Site Structure of Yeast Phosphoglycerate Kinase

Abstract

The enzyme 3‐phosphoglycerate kinase from yeast has been studied by observation of the proton nuclear magnetic resonance spectrum at 270 MHz using Fourier transform techniques. Difference spectroscopy was used to enhance the resolution and to identify specific ligand binding effects and conformational changes. Perturbations involving single protons of amino‐acid residues could thus be detected despite the relatively high molecular weight of the protein (47000), particularly in the aromatic (6–9 ppm) and methylene (2ndash;3 ppm) regions of the spectrum. A. pH titration enabled assignment of the C‐2 and C‐4 imidazole protons of each of three histidine residues remote from the active site, and the determination of their pKa values as 8.20 and 7.05 (two) respectively. The former histidine is deduced to lie buried within the polypeptide chain whilst the latter pair lie in practically identical environments on the surface of the enzyme exposed to solvent. Conformation changes were monitored on binding ADP and ATP both with and without Mg2+. A single site was observed for Mg · ADP, Mg · ATP, La · ATP and the uncomplexed nucleotides. Using paramagnetic difference spectroscopy, the line‐broadening inhibitor Gd · ATP and the substrate Mn · ATP were found to induce identical perturbations of the enzyme resonances, showing that these species bind identically. The shift probes Pr · ATP and Eu · ATP could then be applied to map geometrically the enzyme active site. Three non‐titratable (with pH) histidine resonances, three non‐histidine aromatic resonances and one resonance corresponding to a methylene group of a polar amino acid were located vectorially relative to the ATP unit. No group is close enough to bind directly in the first coordination sphere of the metal. The conformation of lanthanide‐ATP in the active site was effectively unchanged with respect to free lanthanide‐ATP. The conformation changes induced by the nucleotides were further resolved by analysis of the separate effects of adenosine and of lanthanide‐pyrophosphate; the latter caused no conformation change but bound specifically at the nucleotide phosphate binding site. Sulphate anions (which are often used as a medium in growing crystals for X‐ray structure analysis) were shown also to compete for the phosphate groups of the nucleotides. A conformation change was observed on binding 3‐phospho‐D‐glycerate, and a study of the quaternary complexes E · ADP · P‐glycerate · Gd and E · ATP · P‐glycerate · Gd indicated that P‐glycerate, although binding close to the nucleotides, did not significantly perturb the E · metal · nucleotide conformations. Copyright © 1976, Wiley Blackwell. All rights reserved