A comparison of the reactivity and stability of wild type and His388 Gln mutant phosphoglycerate kinase from yeast

Abstract

A variety of physico-chemical techniques have been used to probe the possible interactions between the characteristic structural domains of yeast phosphoglycerate kinase by comparison of the wildtype enzyme with the specific H388Q mutant in which a potential interaction between His388 and Glu190 in the crucial interdomain region is disrupted. Enzyme kinetic studies indicate that, despite being structurally remote from the active site, this mutation has significant effects on both the VmaX and K,,, values for various substrates. The single cysteine residue in the N domain of the protein is markedly more reactive in the mutant, and this enhanced accessibility is moderated by binding of substrates and various anions. Differences are also observed in the near-ultraviolet CD spectra of these proteins. The chemical and thermal stability of the mutant enzyme is reduced, as indicated from guanidinium chloride and differential-scanning calorimetry denaturation studies. Moreover, interdomain interactions seem to be altered in the mutant, resulting in the appearance of independent thermal transition for the two domains, in contrast to the single cooperative transition observed for the wild-type enzyme. The conformational and/or dynamic effects of the mutation on the H388Q enzyme are therefore various and not solely localised in the hinge region.Phosphoglycerate kinase (PGK) catalyses reversible phosphoryl transfer from 1,3-bisphosphoglycerate to ADP in a key ATP-producing step of glycolysis. The enzyme reaction requires metal ions (Mg2+ or Mn'') which form a complex with the nucleotide substrates [I]. PGK isolated from various sources show strong sequence homology and are monomeric proteins with a molecular mass of about 45 kDa. The X-ray structures of the yeast and horse muscle enzymes [2, 31 have shown that the single polypeptide chain is organised into two structurally distinct domains of approximately equal size. In the yeast enzyme the nucleotide and glycerate substrates bind in separate sites close to the interdomain cleft. Mg-ATP or Mg-ADP molecules bind competitively in a shallow depression on the C domain while the most plausible site for 3-phosphoglycerate and 1,3-bisphosphoglycerate is located close to the face of the N domain [2]. In view of the distance between these binding sites (some 1 nm), a hinge-bending model has been proposed for PGK in which movement around the waist region (or hinge) between the two domains expels water and brings the substrates into an orientation and proximity which facilitate phosphoryl transfer [2, 31. Evidence for such a conformational change during catalysis has come from a number of studies including low angle X-ray scattering [4,5] [XI, while consistent with significant conformational change, has not been reproduced elsewhere [9]). Neither the extent nor the possible mechanism of hinge bending during catalysis are known, but the involvement of a His388-Glul90 interaction in the waist region of the enzyme as a mediator of domain closure has been proposed [ZJ. His388 is con...

Site‐directed mutagenesis of proline 204 in the ‘hinge’ region of yeast phosphoglycerate kinase

“…DSC of the wild‐type yeast PGK sample gave results similar to previously published observations on this enzyme [25, 26]. The typical thermogram ( Fig.…”

The effects of temperature on the kinetics and stability of mesophilic and thermophilic 3-phosphoglycerate kinases

“…Due to its hinge-bending mechanism the PGK active site may be more susceptible to limited and localized unfolding before the more gross global unfolding processes become significant. Some evidence for this has been reported during chemical denaturation of yeast [45,46] and horse-muscle PGK [45]. This is an attractive possible reason for the decline in reaction velocity before there is any significant irreversible denaturation, and is not inconsistent with the concept of intermediate I forms.…”

Correlation between conformational stability of the ternary enzyme–substrate complex and domain closure of 3‐phosphoglycerate kinase

“…Separated DSC transitions attributable to the individual PGK domains have been observed in the case of engineered mutants of yeast PGK that contain modifications in the hinge region between the two domains [20,68] and in the case of cold‐active PGK [76], possibly due to weaker interdomain interactions in these cases. It is notable that the substrate 3‐phospho‐ d ‐glycerate (3‐PG) in one case [68], while 3‐PG and MgADP together in the other case [76] caused merging of the two transitions into a single one. This indicates increased domain cooperativity upon substrate binding in the mutants.…”

“…Heat-or cold-induced unfolding of yeast [19,20,[64][65][66][67][68][69][70][71][72][73], thermophile [64,65,74,75] and cold-active [76] PGKs in the absence [64][65][66][67][68][69][70][71][72][73][74][75] or presence [19,20,76] of substrates has been monitored by differential scanning calorimetry (DSC), a widely used approach to determine the number of folding units within a protein molecule. Disruption of native PGK structure upon cooling occurs in two distinct stages, corresponding to independent and reversible unfolding of the individual domains [66,[70][71][72].…”