Anion Binding to Yeast Phosphoglycerate Kinase

Anion binding to yeast phosphoglycerate kinase has been investigated using 'H-NMR spectroscopy. The use of anionic paramagnetic probes, [Cr(CN), ,I3-, has enabled the location of the primary anion binding site in the 'basic-patch' region of the amino-terminal domain. The anions interact most closely with Arg-65 and Arg-168. The binding of these and a variety of other anions to this site is directly competitive with the binding of the substrate, 3-phosphoglycerate. Binding of 3-phosphoglycerate and 1,3-bisphosphoglycerate is, however, stronger than expected on the basis of anionic charge and causes conformational changes in the protein not seen with any of the other simple spherical anions investigated. This must be part, at least, of the substrate specificity.Evidence for a secondary anion binding site involving the side chains of surface lysine residues is also presented. It is suggested that the primary anion site is responsible for the observed activation by anions at low concentrations wbile the secondary site leads to inhibition at higher anion concentrations. The kinetics fit these deductions and a scheme for kinase activity is presented.There are several features in the reaction catalysed by phosphoglycerate kinase (PGK) between ATP and 3-phosphoglycerate (3-P-glycerate) which are little understood. (a) Although it is known with reasonable precision where ATP binds, the binding of 3-P-glycerate remains ill-defined [I, 21. Several experiments, however, have shown the 3-P-glycerate binding site to be associated with a basic patch of residues in the N-terminal domain [3-51 (see Fig. 7). Such a site was postulated from earlier crystallographic studies of horsemuscle PGK [I], but is not in total agreement with the crystallographically determined site of yeast PGK [2]. (b) Crystallographic studies have also led to the suggestion that a 'hingebending' conformational change involving helix rotations and the relative movements of the two large domains is necessary for catalysis [I, 61. Recent NMR results where phosphate transfer takes place in the catalytic reaction.' The activating effect of sulphate at lower concentrations and the substrate activation phenomena [12] were interpreted in terms of a two-step dissociation of 1,3-bisphosphoglycerate (1 ,3-P2-glycerate) [I I]. In effect the general anion site is the same as, or close to, the 3-P-glycerate binding site and at lower concentrations the anion activates the enzyme by acceleration of the dissociation rate of the product, 1,3-P2-glycerate, while at higher concentrations the dominant effects is inhibition through competition with the initial substrate binding. , however, have suggested the existence of at least two sulphate-binding sites, the strongest being a general anion site and the other being the catalytic centre. Evidence for weaker secondary anion binding has also come from previous NMR studies of yeast PGK [7, 131. Wrobel and Stinson [I41 labelled the single thiol of yeast PGK (Cys-97 situated z 2.5 nm from the crystallographically determined 3-...

show abstract

“…As noted by Wrobel and Stinson [14], there is a linear relationship between the negative logarithm of the Kd and the anionic charge (Fig. 3).…”

Section: Titration Of Pgk With Anionsmentioning

confidence: 87%

“…These authors [14] put forward several arguments suggesting that the anion site they were monitoring was not in fact the active site. However, since only one site was found in gelfiltration studies, Scopes [Ill suggested that they must be looking at the active site, though indirectly.…”

mentioning

confidence: 99%

An NMR study of anion binding to yeast phosphoglycerate kinase

Fairbrother¹,

GRAHAM²,

Williams³

1990

European Journal of Biochemistry

View full text Add to dashboard Cite

show abstract

“…Since submitting this manuscript, a paper on anion binding to yeast phosphoglycerate kinase [26] has been published. Many of the findings are similar to those reported here.…”

Section: Discussionmentioning

confidence: 99%

Binding of Substrates and Other Anions to Yeast Phosphoglycerate Kinase

Scopes

1978

Eur J Biochem

View full text Add to dashboard Cite

1. The binding of all four substrates to yeast phosphoglycerate kinase has been studied using a gel filtration technique. The binding of phosphate and sulphate anions has also been investigated.2. Two sites for each adenine nucleotide were found, one site being weaker than the other by between 30 and 50-fold. Only one binding site for the phosphoglycerate substrates was found.3. 1,3-Bisphosphoglycerate (1 ,3-P2-glycerate) bound to the enzyme approximately 1000 times tighter than the other three substrates, its dissociation constant being 0.06 pM at ionic strength 0.15 M.4. Sulphate and phosphate were mutually competitive and sulphate competed with the binding of all substrates except MgADP. MgADP bound to the enzyme more weakly in the presence of sulphate. The dissociation constant for sulphate binding was 1.6 mM at ionic strength of 0.15 M, and 0.05 mM at ionic strength 0.015 M.5. These results are consistent with sulphate acting as a competitive inhibitor, as found by kinetic studies at high sulphate concentrations. The activatory effect of sulphate at lower concentrations and the substrate activation phenomena displayed by this enzyme, are interpreted in terms of a two-step dissociation of 1 ,3-P2-glycerate. The presence of moderate concentrations of MgATP, 3-phosphoglycerate or sulphate causes acceleration of the rate of dissociation of the product, 1,3-P2-glycerate, this being the rate-limiting step in the overall enzyme reaction.

show abstract

“…Typically 5 ml solution, in two Amicon Centricon 30 tubes, was reduced to a volume of 200 pl. The sulphate, which affects enzyme activity [14,[16][17][18][19], was removed by washing with 3 -4 vol. ('H,)acetate buffer.…”

Section: Methodsmentioning

confidence: 99%

NMR analysis of the interdomain region of yeast phosphoglycerate kinase

Wilson

Williams

Littlechild

et al. 1988

European Journal of Biochemistry

View full text Add to dashboard Cite

Previous proton and phosphorus nuclear magnetic resonance studies with yeast phosphoglycerate kinase have been extended using a higher-resolution spectrometer and a greater variety of binding agents. The new study shows that, apart from a few isolated mobile side chains distributed over the protein surface, there is a mobile section of phosphoglycerate kinase associated with the inter-domain region of the molecule. This region gives relatively well resolved resonances which are quite distinct from those originating from the remainder of the protein. This suggests that the molecule fluctuates between many states including several open or substrate binding forms in addition to the closed and supposedly catalytically competent form of the enzyme. The occupancy of these states appears to be affected by several anions including sulphate, phosphate and cobalticyanide, as well as substrates and their analogues.Low-angle X-ray scattering studies carried out with yeast phosphoglycerate kinase (PGK) have shown that a reduction of more than 0.1 nm in the enzyme's radius of gyration occurs following the formation of a true ternary complex [l]. This finding, taken together with the marked bilobal nature of the molecule [2, 31, has led to the conclusion that at least two markedly different structural forms of this enzyme exist during substrate conversion. Crystallographic studies of the enzymes from both yeast In this paper we expand on our previous analysis [6] of yeast PGK by proton and phosphorous nuclear magnetic resonance (NMR) methods. Features of the earlier NMR spectra provided information concerning the relationship between the binding sites for the adenine nucleotides and for the phosphoglycerates. In the absence of a sequence for, and of structural data relating to, the closed and therefore catalytically competent form of the enzyme, it was possible to describe the phosphoglycerate binding site in terms of three histidine residues in close proximity to the phosphate binding sites of the bound nucleotide substrates. This observation is entirely compatible with subsequent model building studies (see Fig. 1) on the assumption that the NMR measurements relate to a distribution between the closed and open structures [7]. The earlier NMR data showed that conformational changes occurred on binding substrates and inhibitors [6]. In the case of large proteins, NMR methods are likely to remain restricted by the inability to resolve resonances which will inevitably be relatively broad. However experience has shown that even large proteins often show some narrow lines in their spectra and, although a few arise from isolated mobile single residues, it is generally believed that these lines relate to more mobile segments of the proteins [8 -121. Naturally these regions are likely to be more exposed than buried. When recording the 500-MHz proton NMR spectrum of PGK it was noted that there was such a set of narrower lines lying on top of a broad spectrum. In this paper we describe the way in which we have set about relating sidech...

show abstract

Anion Binding to Yeast Phosphoglycerate Kinase

Cited by 32 publications

References 19 publications

An NMR study of anion binding to yeast phosphoglycerate kinase

An NMR study of anion binding to yeast phosphoglycerate kinase

Binding of Substrates and Other Anions to Yeast Phosphoglycerate Kinase

NMR analysis of the interdomain region of yeast phosphoglycerate kinase

Contact Info

Product

Resources

About