Characterisation of yeast phosphoglycerate kinase modified by mutagenesis at residue 21

Abstract

Site-directed mutagenesis has been used to produce mutant forms of yeast phosphoglycerate kinase in which the conserved active-site residue, Arg21, has been replaced by a methionine or a lysine. Kinetic results obtained using these mutant enzymes show that their K,,, for both 3-phospho-D-glycerate and ATP are significantly different from those recorded for the wild-type enzyme. The V,,, for the lysine mutant is reduced by a factor of two from that of the wild-type enzyme whereas the V,,, for the methionine mutant is reduced more than sevenfold. A very clean electron-densitydifference map shows little, if any, evidence of a structural change associated with the C-terminal domain, although resonances in the NMR spectra associated with the ATP-binding site (C-terminal domain) are also affected by the mutation as one might expect from the kinetic results. The NMR data show that binding at both the 3-phospho-~-glycerate and the non-productive ATP-binding site (associated with the N-terminal domain) are affected in the mutant in a way which is different to that associated with the wild-type enzyme. These results, taken together with the X-ray and kinetic data, indicate that the non-productive ATP-binding site and the activating anion-binding site are both associated with the basic patch region of yeast phosphoglycerate kinase.The glycolytic enzyme phosphoglycerate kinase (PGK) has been isolated and the amino acid sequencje obtained from over 25 different species [l]. Tertiary structures have also been determined for both eukaryote (yeast [2, 31 and horse [4, 51) and prokaryotic (Bucillus steurothermophilus, unpublished results) enzymes. All three enzymes are markedly bilobal and show similar domain orientations. A wealth of structural information is also available from 'H-NMR studies of the yeast enzyme [6 -111.All the available sequence and structural data indicates that there is a characteristic cluster of basic residues associated with the N-terminal domain facing the nucleotide-binding site. This region of the molecule contains five arginine residues (21, 38, 65, 121 and 168: yeast numbering) that are totally conserved in all PGK sequenced (Fig. 1). This so called basicpatch region also contains two totally conserved histidine residues (62 and 167: yeast numbering). As part of an extensive study of enzyme structure and function, we have embarked on a programme aimed at substituting these conserved residues by site-directed mutagenesis [12, 131. In order to characterise the activity of this ubiquitous enzyme, each site-specific mutant will be studied using kinetic, crystallographic and 'H-NMR techniques. Here we report on the preparation and kinetic properties of two specific mutations of Arg21 (yeast enzyme). Emphasis is given to the importance of using both crystallographic (solid state) and NMR (solution state) data when attempting to interpret kinetic data derived from even relatively simple enzyme systems. MATERIALS AND METHODSWild-type PGK and the two mutant enzymes, [R21K]-PGK and [R21M]PGK, were prepared ...

Substrate-Assisted Movement of the Catalytic Lys 215 during Domain Closure: Site-Directed Mutagenesis Studies of Human 3-Phosphoglycerate Kinase

“…3-PG is bound nearly in the same way in all PGK crystal structures. In agreement, site-directed mutagenesis studies (24)(25)(26)(38)(39)(40)(41)(42)(43) have confirmed the importance of the basic residues at the inner surface of the N-domain in binding of this substrate. The extended basic patch can also bind various anions, as shown by various techniques (42,(44)(45)(46)(47).…”

“…These activation phenomena are peculiar regulatory behaviors of PGK, the molecular basis of which is still not completely understood. The anion activation is diminished or completely abolished in the case of some basic patch mutants (39,41,43), but the data have not yet provided a satisfactory explanation about the location of the activating site and the molecular mechanism of activation. A hypothesis was put forward about formation of the activating anionic site only upon domain closure (50).…”

“…To test the multiple roles of Lys 215 (mammalian PGK numbering) in this work, we have carried out site-directed mutagenesis studies on hPGK. Although no 3D structure of hPGK has been determined, its primary sequence is identical (up to 98%) to that of pig muscle PGK, for which various crystal structures ( 24−26 , − are known. Thus, Lys 215 was replaced with either the neutral Ala or the similarly charged Arg, and the mutants (K215A and K215R) were characterized in enzyme kinetic and substrate binding studies.…”

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

“…The apparent anomalous increase in activity of the Pro204Phe mutant may be due to restructuring of the enzyme in sulphate. It is accepted that the ‘basic patch’ is an extended anion binding site containing both the triose binding site and the anion activatory site [30, 31]. The higher affinity of the activatory site allows anions to bind at low anionic concentrations, whilst anions only bind the inhibitory site at higher anionic concentrations.…”

Transient and Equilibrium Kinetic Studies on Yeast 3-Phosphoglycerate Kinase. Evidence That an Intermediate Containing 1,3-Bisphosphoglycerate Accumulates in the Steady State

“…Watson et al, 1982) and by NMR [reviewed in Joao and Williams (1993)]. PGK is a single polypeptide chain of about 45 kDa, and the probing of the roles of several amino acid residues in its catalytic function has been carried out by site-directed mutagenesis [e.g., Ballery et al (1990), Sherman et al (1992), andWalker et al (1992); reviewed in Joao and Williams (1993)]. From these structural studies the binding of either substrate induces a conformational change, that with PG being the larger.…”