`In crystallo' substrate binding triggers major domain movements and reveals magnesium as a co‐activator of Trypanosoma brucei pyruvate kinase. (25th September 2013)

• Record Type: Journal Article
• Authors: Zhong, Wenhe; Morgan, Hugh P.; McNae, Iain W.; Michels, Paul A. M.; Fothergill‐Gilmore, Linda A.; Walkinshaw, Malcolm D.
• Is Part Of: Acta crystallographica. Volume 69:Part 9(2013:Sep.)
• Page Start: 1768
• Page End: 1779
• Publication Date: 2013-09-25
• Subjects: Biomolecules -- Structure -- Periodicals; Physical biochemistry -- Periodicals; X-ray crystallography -- Periodicals; Crystallography -- Periodicals; 572
• DOI: 10.1107/S0907444913013875
• Languages: English
• ISSNs: 0907-4449

Abstract

<abstract abstract-type="main" xml:lang="en"> <title> <x xml:space="preserve">Abstract</x> </title> <p>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 <italic>Trypanosoma brucei</italic>. PEP soaked into crystals of the enzyme with bound allosteric activator fructose 2, 6‐bisphosphate (F26BP) and Mg<sup>2+</sup> triggers a substantial 23° rotation of the B domain `<italic>in crystallo</italic>', 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.</p> </abstract>