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`In crystallo' substrate binding triggers major domain movements and reveals magnesium as a co-activator of Trypanosoma brucei pyruvate kinase.

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Zhong, Wenhe Morgan, Hugh P McNae, Iain W Michels, Paul A M [UCL] Fothergill-Gilmore, Linda A Walkinshaw, Malcolm D

The active site of pyruvate kinase (PYK) is located between the AC core of the enzyme and a mobile lid corresponding to domain B. Many PYK structures have already been determined, but the first `effector-only' structure and the first with PEP (the true natural substrate) are now reported for the enzyme from Trypanosoma brucei. PEP soaked into crystals of the enzyme with bound allosteric activator fructose 2,6-bisphosphate (F26BP) and Mg(2+) triggers a substantial 23° rotation of the B domain `in crystallo', resulting in a partially closed active site. The interplay of side chains with Mg(2+) and PEP may explain the mechanism of the domain movement. Furthermore, it is apparent that when F26BP is present but PEP is absent Mg(2+) occupies a position that is distinct from the two canonical Mg(2+)-binding sites at the active site. This third site is adjacent to the active site and involves the same amino-acid side chains as in canonical site 1 but in altered orientations. Site 3 acts to sequester Mg(2+) in a `priming' position such that the enzyme is maintained in its R-state conformation. In this way, Mg(2+) cooperates with F26BP to ensure that the enzyme is in a conformation that has a high affinity for the substrate.

Document type Article de périodique (Journal article) – Article de recherche
Access type Accès libre
Publication date 2013
Language Anglais
Journal information "Acta crystallographica. Section D, Biological crystallography" - Vol. 69, no.9, p. 1768-1779 (2013)
Peer reviewed yes
Publisher Wiley-Blackwell Munksgaard ((United States) [S.l.])
issn 0907-4449
e-issn 1399-0047
Publication status Publié
Affiliation UCL - SSS/DDUV - Institut de Duve
Links
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Bibliographic reference Zhong, Wenhe ; Morgan, Hugh P ; McNae, Iain W ; Michels, Paul A M ; Fothergill-Gilmore, Linda A ; et. al. `In crystallo' substrate binding triggers major domain movements and reveals magnesium as a co-activator of Trypanosoma brucei pyruvate kinase.. In: Acta crystallographica. Section D, Biological crystallography, Vol. 69, no.9, p. 1768-1779 (2013)
Permanent URL http://hdl.handle.net/2078.1/134134