Anion Activation of 3-Phosphoglycerate Kinase Requires Domain Closure

Szilágyi, Andrea N.; Vas, Mária

doi:10.1021/bi985048s

Cited by 4 publications

(4 citation statements)

References 0 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The fit of the linear regions of the plots is of initial rates (υ 0 ) versus initial substrate concentrations ([S] 0 ). The slopes were used to calculate k cat /K M values since PGK is activated by the substrate anion and therefore displays complex kinetics [47].The k cat /K M for wild type PGK was in disagreement with previously reported measurements [18].…”

Section: Supporting Informationmentioning

confidence: 67%

Mechanisms of Protein Sequence Divergence and Incompatibility

2013

View full text Add to dashboard Cite

Alignments of orthologous protein sequences convey a complex picture. Some positions are utterly conserved whilst others have diverged to variable degrees. Amongst the latter, many are non-exchangeable between extant sequences. How do functionally critical and highly conserved residues diverge? Why and how did these exchanges become incompatible within contemporary sequences? Our model is phosphoglycerate kinase (PGK), where lysine 219 is an essential active-site residue completely conserved throughout Eukaryota and Bacteria, and serine is found only in archaeal PGKs. Contemporary sequences tested exhibited complete loss of function upon exchanges at 219. However, a directed evolution experiment revealed that two mutations were sufficient for human PGK to become functional with serine at position 219. These two mutations made position 219 permissive not only for serine and lysine, but also to a range of other amino acids seen in archaeal PGKs. The identified trajectories that enabled exchanges at 219 show marked sign epistasis - a relatively small loss of function with respect to one amino acid (lysine) versus a large gain with another (serine, and other amino acids). Our findings support the view that, as theoretically described, the trajectories underlining the divergence of critical positions are dominated by sign epistatic interactions. Such trajectories are an outcome of rare mutational combinations. Nonetheless, as suggested by the laboratory enabled K219S exchange, given enough time and variability in selection levels, even utterly conserved and functionally essential residues may change.

show abstract

Section: Supporting Informationmentioning

confidence: 67%

Mechanisms of Protein Sequence Divergence and Incompatibility

2013

View full text Add to dashboard Cite

show abstract

“…The activity of hPGK was measured as described earlier [10]. For the reaction with the substrates 3-PG and MgATP the rate equation describing activation by the excess of the substrate [12] was used for analysis of the data. Activity measurements with the substrates 1,3-BPG and MgADP were analysed using the Michaelis-Menten equation.…”

Section: Enzyme Kinetic Studiesmentioning

confidence: 99%

Role of side‐chains in the operation of the main molecular hinge of 3‐phosphoglycerate kinase

et al. 2008

Self Cite

View full text Add to dashboard Cite

The single mutants (F165A, E192A, F196A, S392A, T393A) at and near the main hinge (b-strand L) of human 3-phosphoglycerate kinase (hPGK) exhibit variously reduced enzyme activity, indicating the cumulative effects of these residues in regulating domain movements. The residues F165 and E192 are also essential in maintaining the conformational integrity of the whole molecule, including the hinge-region. Shortening of bL by deleting T393 has led to a dramatic activity loss and the concomitant absence of domain closure (as detected by small angle X-ray scattering), demonstrating the role of bL in functioning of hPGK. The role of each residue in the conformational transmission is described.

show abstract

“…[26,31] Due to this fact, PGK has become one of the model systems to study properties of multi-domain protein folding. [33][34][35] In this respect the effect of domain interactions on folding rates and the succession of specific folding events are of particular interest. In order to elucidate details of the folding or unfolding pathways, multiple intramolecular distances typically have to be analyzed.…”

Section: Introductionmentioning

confidence: 99%

Native and Unfolded States of Phosphoglycerate Kinase Studied by Single‐Molecule FRET

Rosenkranz¹,

Schlesinger²,

Gabba³

et al. 2010

ChemPhysChem

View full text Add to dashboard Cite

Single-molecule Förster resonance energy transfer (FRET) measurements with phosphoglycerate kinase from yeast were performed at different concentrations of guanidine hydrochloride. From these steady-state measurements we obtained FRET efficiency histograms characterizing structural properties of individual proteins at different stages between the native and the fully unfolded state. Native proteins exhibit a slightly more expanded structure under buffer conditions without denaturant as compared to conditions with denaturant. At 0.5 M GndHCl an unfolded state population that exhibits a significantly expanded structure as compared to the native state, emerges. The unfolded state is characterized by a pronounced broadening of the efficiency distribution, which indicates a large structural and/or dynamical heterogeneity within the population. At high denaturant concentrations, well above the unfolding transition at C(1/2)~0.7 M, we observe a progressive expansion of the protein structure, namely globule-coil transition.

show abstract

Anion Activation of 3-Phosphoglycerate Kinase Requires Domain Closure

Cited by 4 publications

References 0 publications

Mechanisms of Protein Sequence Divergence and Incompatibility

Mechanisms of Protein Sequence Divergence and Incompatibility

Role of side‐chains in the operation of the main molecular hinge of 3‐phosphoglycerate kinase

Native and Unfolded States of Phosphoglycerate Kinase Studied by Single‐Molecule FRET

Contact Info

Product

Resources

About