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J Biol Chem. 2016 Apr 22;291(17):9244-56. doi: 10.1074/jbc.M115.676270. Epub 2016 Feb 15.

Conformational Dynamics and Allostery in Pyruvate Kinase.

Author information

1
From the Biomolecular Interaction Centre and School of Biological Sciences, University of Canterbury, Private Bag 4800, Christchurch 8041, New Zealand.
2
Department of Chemistry, York University, Toronto, Ontario M3J 1P3, Canada.
3
Biology and Biochemistry, University of Houston, Houston, Texas 77204.
4
Department of Chemistry, York University, Toronto, Ontario M3J 1P3, Canada, Centre for Research in Mass Spectrometry, Department of Chemistry, York University, 4700 Keele Street, Toronto, Ontario M3J 1P3, Canada, and dkwilson@yorku.ca.
5
From the Biomolecular Interaction Centre and School of Biological Sciences, University of Canterbury, Private Bag 4800, Christchurch 8041, New Zealand, Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, 30 Flemington Road, Parkville, Victoria 3010, Australia renwick.dobson@canterbury.ac.nz.

Abstract

Pyruvate kinase catalyzes the final step in glycolysis and is allosterically regulated to control flux through the pathway. Two models are proposed to explain how Escherichia coli pyruvate kinase type 1 is allosterically regulated: the "domain rotation model" suggests that both the domains within the monomer and the monomers within the tetramer reorient with respect to one another; the "rigid body reorientation model" proposes only a reorientation of the monomers within the tetramer causing rigidification of the active site. To test these hypotheses and elucidate the conformational and dynamic changes that drive allostery, we performed time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange studies followed by mutagenic analysis to test the activation mechanism. Global exchange experiments, supported by thermostability studies, demonstrate that fructose 1,6-bisphosphate binding to the allosteric domain causes a shift toward a globally more dynamic ensemble of conformations. Mapping deuterium exchange to peptides within the enzyme highlight site-specific regions with altered conformational dynamics, many of which increase in conformational flexibility. Based upon these and mutagenic studies, we propose an allosteric mechanism whereby the binding of fructose 1,6-bisphosphate destabilizes an α-helix that bridges the allosteric and active site domains within the monomeric unit. This destabilizes the β-strands within the (β/α)8-barrel domain and the linked active site loops that are responsible for substrate binding. Our data are consistent with the domain rotation model but inconsistent with the rigid body reorientation model given the increased flexibility at the interdomain interface, and we can for the first time explain how fructose 1,6-bisphosphate affects the active site.

KEYWORDS:

Escherichia coli (E. coli); allosteric regulation; conformational flexibility; fructose 1,6-bisphosphate; glycolysis; hydrogen-deuterium exchange; pyruvate kinase

PMID:
26879751
PMCID:
PMC4861489
DOI:
10.1074/jbc.M115.676270
[Indexed for MEDLINE]
Free PMC Article

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