Flexibility and folding of phosphoglycerate kinase.
Abstract: Flexibility and folding of phosphoglycerate kinase, a two-domain monomeric enzyme, have been studied using a wide variety of methods including theoretical approaches. Mutants of yeast phosphoglycerate kinase have been prepared in order to introduce cysteinyl residues as local probes throughout the molecule without perturbating significantly the structural or the functional properties of the enzyme. The apparent reactivity of a unique cysteine in each mutant has been used to study the flexibility of PGK. The regions of larger mobility have been found around residue 183 on segment beta F in the N-domain and residue 376 on helix XII in the C-domain. These regions are also parts of the molecule which unfold first. Ligand binding induces conformational motions in the molecule, especially in the regions located in the cleft. Moreover, the results obtained by introducing a fluorescent probe covalently linked to a cysteine are in agreement with the helix scissor motion of helices 7 and 14 assumed by Blake to direct the hinge bending motion of the domains during the catalytic cycle. The folding process of both horse muscle and yeast phosphoglycerate kinases involves intermediates. These intermediates are more stable in the horse muscle than in the yeast enzyme. In both enzymes, domains behave as structural modules capable of folding and stabilizing independently, but in the horse muscle enzyme the C-domain is more stable and refolds prior to the N-domain, contrary to that which has been observed in the yeast enzyme. A direct demonstration of the independence of domains in yeast phosphoglycerate kinase has been provided following the obtention of separated domains by site-directed mutagenesis. These domains have a native-like structure and refold spontaneously after denaturation by guanidine hydrochloride.
Publication Date: 1990-06-01 PubMed ID: 2124145DOI: 10.1016/0300-9084(90)90066-pGoogle Scholar: Lookup
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Summary
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The research focuses on the flexibility and folding of phosphoglycerate kinase, a two-domain monomeric enzyme, using different methods including theoretical approaches and mutants of yeast phosphoglycerate kinase.
Methodology
- The study adopts various techniques for examining the flexibility and folding of phosphoglycerate kinase. One of the main methods used is introducing mutants of yeast phosphoglycerate kinase, which enable the introduction of cysteinyl residues as local probes throughout the enzyme molecule.
- The researchers ensured these probes did not majorly disturb the enzyme’s structural or functional properties. The apparent reactivity of a unique cysteine in each mutant has been used to study the flexibility of phosphoglycerate kinase.
Findings
- The research identified regions of highest mobility within the enzyme, specifically residue 183 on segment beta F in the N-domain and residue 376 on helix XII in the C-domain. These areas were also found to unfold first in the structure of the enzyme.
- The team discovered that ligand binding triggers a variety of conformational motions in the enzyme, with the most activity in regions located in the ‘cleft’ of the enzyme structure.
- The results obtained from a fluorescent probe linked to cysteine aligned with the hypothesized motion of helices 7 and 14, suggesting this ‘helix scissor’ motion drives the hinge bending motion of the domains throughout the catalytic cycle.
Folding Process of Phosphoglycerate Kinase
- The study also analyzed the folding processes for both horse muscle and yeast phosphoglycerate kinases and found both involve the use of intermediates.
- These intermediates were noted to be more stable in horse muscle than in yeast phosphoglycerate kinase.
- For both enzymes, the domains behaved as structural modules that can fold and stabilize independently. However, in horse muscle enzyme, the C-domain has shown higher stability and refolds before the N-domain, which is opposite to what takes place in the yeast enzyme.
- The researchers achieved a direct confirmation of the independence of domains in yeast phosphoglycerate kinase by obtaining separated domains using site-directed mutagenesis. These separated domains displayed a native-like structure and were found to refold spontaneously after denaturation by guanidine hydrochloride.
Cite This Article
APA
Yon JM, Desmadril M, Betton JM, Minard P, Ballery N, Missiakas D, Gaillard-Miran S, Perahia D, Mouawad L.
(1990).
Flexibility and folding of phosphoglycerate kinase.
Biochimie, 72(6-7), 417-429.
https://doi.org/10.1016/0300-9084(90)90066-p Publication
Researcher Affiliations
- Laboratoire d'enzymologie physico-chimique et moléculaire, Unité de Recherche du CNRS, associée à l'Université de Paris-Sud, Orsay, France.
MeSH Terms
- Animals
- Binding Sites
- Horses
- Models, Molecular
- Mutagenesis, Site-Directed
- Phosphoglycerate Kinase / chemistry
- Phosphoglycerate Kinase / genetics
- Phosphoglycerate Kinase / metabolism
- Protein Conformation
- Protein Denaturation
- Saccharomyces cerevisiae / enzymology
- Saccharomyces cerevisiae / genetics
Citations
This article has been cited 6 times.- Sechovcová H, Kulhavá L, Fliegerová K, Trundová M, Morais D, Mrázek J, Kopečný J. Comparison of enzymatic activities and proteomic profiles of Butyrivibrio fibrisolvens grown on different carbon sources.. Proteome Sci 2019;17:2.
- Chen C, Long L, Zhang F, Chen Q, Chen C, Yu X, Liu Q, Bao J, Long Z. Antifungal activity, main active components and mechanism of Curcuma longa extract against Fusarium graminearum.. PLoS One 2018;13(3):e0194284.
- Valentini G, Maggi M, Pey AL. Protein Stability, Folding and Misfolding in Human PGK1 Deficiency.. Biomolecules 2013 Dec 18;3(4):1030-52.
- Zheng H, Filippova EV, Tkaczuk KL, Dworzynski P, Chruszcz M, Porebski PJ, Wawrzak Z, Onopriyenko O, Kudritska M, Grimshaw S, Savchenko A, Anderson WF, Minor W. Crystal structures of putative phosphoglycerate kinases from B. anthracis and C. jejuni.. J Struct Funct Genomics 2012 Mar;13(1):15-26.
- Thomas TM, Scopes RK. The effects of temperature on the kinetics and stability of mesophilic and thermophilic 3-phosphoglycerate kinases.. Biochem J 1998 Mar 15;330 ( Pt 3)(Pt 3):1087-95.
- Root DD, Reisler E. The accessibility of etheno-nucleotides to collisional quenchers and the nucleotide cleft in G- and F-actin.. Protein Sci 1992 Aug;1(8):1014-22.
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