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

DOI: 10.1107/S0907444913013875 DOI Help
PMID: 23999300 PMID Help

Authors: Wenhe Zhong (University of Edinburgh) , Hugh Morgan (University of Edinburgh) , Iain Mcnae (University of Edinburgh) , Paul A.m. Michels (The University of Edinburgh) , Linda Fothergill-gilmore (University of Edinburgh) , Malcolm Walkinshaw (University of Edinburgh)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Acta Crystallographica Section D Biological Crystallography , VOL 69 , PAGES 1768 - 1779

State: Published (Approved)
Published: May 2013

Abstract: 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 Mg2+ triggers a substantial 23° rotation of the B domain `in crystallo', resulting in a partially closed active site. The interplay of side chains with Mg2+ and PEP may explain the mechanism of the domain movement. Furthermore, it is apparent that when F26BP is present but PEP is absent Mg2+ occupies a position that is distinct from the two canonical Mg2+-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 Mg2+ in a `priming' position such that the enzyme is maintained in its R-state conformation. In this way, Mg2+ cooperates with F26BP to ensure that the enzyme is in a conformation that has a high affinity for the substrate.

Journal Keywords: Crystallography; X-Ray; Fructosediphosphates; Magnesium; Protein; Tertiary; Pyruvate; Rotation; Substrate; Trypanosoma brucei brucei

Subject Areas: Biology and Bio-materials, Medicine

Instruments: I04-Macromolecular Crystallography , I24-Microfocus Macromolecular Crystallography