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Modulation of a pre-existing conformational equilibrium tunes adenylate kinase activity
Umeå University, Faculty of Science and Technology, Department of Chemistry.
Umeå University, Faculty of Science and Technology, Department of Chemistry.
2012 (English)In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 134, no 40, 16562-16570 p.Article in journal (Refereed) Published
Abstract [en]

Structural plasticity is often required for distinct microscopic steps during enzymatic reaction cycles. Adenylate kinase from Escherichia coli (AKeco) populates two major conformations in solution; the open (inactive) and closed (active) state, and the overall turn-over rate is inversely proportional to the life-time of the active conformation. Therefore, structural plasticity is intimately cou-pled to enzymatic turn-over in AKeco. Here we probe the open to closed conformational equilibrium in the absence of bound substrate with NMR spectroscopy and molecular dynamics simulations. The conformational equilibrium in absence of substrate, and in turn, the turn-over number can be modulated with mutational- and osmolyte-driven perturbations. Removal of one hydrogen bond between the ATP and AMP binding sub-domains results in a population shift towards the open conformation and a resulting increase of kcat. Addition of the osmolyte TMAO to AKeco results in population shift towards the closed conformation and a significant reduction of kcat. The Michaelis constants (KM) scale with the change in kcat, which follows from the influence of the population of the closed conformation for substrate binding affinity. Hence, kcat and KM are mutually dependent and, in the case of AKeco, any perturbation that modulates kcat is mirrored with a proportional response in KM. Thus, our results demonstrate that the equilibrium constant of a pre-existing conformational equilibrium directly affect enzymatic catalysis. From an evolutionary perspective our findings suggests that, for AKeco, there exists ample flexibility to obtain a specificity constant (kcat/KM) that commensurate with the exerted cellular selective pressure.

Place, publisher, year, edition, pages
Washington: American Chemical Society (ACS), 2012. Vol. 134, no 40, 16562-16570 p.
Keyword [en]
Adenylate kinase, enzyme, activity, pre-existing equilibrium, conformational dynamics, osmolyte, NMR
National Category
Chemical Sciences
URN: urn:nbn:se:umu:diva-59505DOI: 10.1021/ja3032482ISI: 000309566400030PubMedID: 22963267OAI: diva2:552794
Available from: 2012-09-17 Created: 2012-09-17 Last updated: 2012-11-16Bibliographically approved

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Ådén, JörgenWolf-Watz, Magnus
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