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  Substrate-Based Allosteric Regulation of a Homodimeric Enzyme

Mehrabi, P., Pietrantonio, C. D., Kim, T. H., Sljoka, A., Taverner, K., Ing, C., et al. (2019). Substrate-Based Allosteric Regulation of a Homodimeric Enzyme. Journal of the American Chemical Society, 141(29), 11540-11556. doi:10.1021/jacs.9b03703.

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https://dx.doi.org/10.1021/jacs.9b03703 (Publisher version)


Mehrabi, P.1, 2, 3, Author              
Pietrantonio, C. D.4, Author
Kim, T. H.4, Author
Sljoka, A.4, 5, 6, Author
Taverner, K.4, Author
Ing, C.7, 8, Author
Kruglyak, N.3, 8, Author
Pomès, R.7, 8, Author
Pai, E. F.1, 3, 8, Author
Prosser, R. S.1, 4, 8, Author
1Department of Medical Biophysics, University of Toronto, ou_persistent22              
2Miller Group, Atomically Resolved Dynamics Department, Max Planck Institute for the Structure and Dynamics of Matter, Max Planck Society, ou_1938288              
3Campbell Family Institute for Cancer Research, Princess Margaret Cancer Centre, Toronto, ou_persistent22              
4Department of Chemistry, University of Toronto, ou_persistent22              
5CREST, Japan Science and Technology Agency (JST), Department of Informatics, School of Science and Technology, Kwansei Gakuin University, ou_persistent22              
6Center for Advanced Intelligence Project, RIKEN, ou_persistent22              
7Program in Molecular Medicine, Research Institute, The Hospital for Sick Children, Toronto, ou_persistent22              
8Department of Biochemistry, University of Toronto, ou_persistent22              


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 Abstract: Many enzymes operate through half-of-the sites reactivity wherein a single protomer is catalytically engaged at one time. In the case of the homodimeric enzyme, fluoroacetate dehalogenase, substrate binding triggers closing of a regulatory cap domain in the empty protomer, preventing substrate access to the remaining active site. However, the empty protomer serves a critical role by acquiring more disorder upon substrate binding, thereby entropically favoring the forward reaction. Empty protomer dynamics are also allosterically coupled to the bound protomer, driving conformational exchange at the active site and progress along the reaction coordinate. Here, we show that at high concentrations, a second substrate binds along the substrate-access channel of the occupied protomer, thereby dampening interprotomer dynamics and inhibiting catalysis. While a mutation (K152I) abrogates second site binding and removes inhibitory effects, it also precipitously lowers the maximum catalytic rate, implying a role for the allosteric pocket at low substrate concentrations, where only a single substrate engages the enzyme at one time. We show that this outer pocket first desolvates the substrate, whereupon it is deposited in the active site. Substrate binding to the active site then triggers the empty outer pocket to serve as an interprotomer allosteric conduit, enabling enhanced dynamics and sampling of activation states needed for catalysis. These allosteric networks and the ensuing changes resulting from second substrate binding are delineated using rigidity-based allosteric transmission theory and validated by nuclear magnetic resonance and functional studies. The results illustrate the role of dynamics along allosteric networks in facilitating function.


Language(s): eng - English
 Dates: 2019-04-062019-06-122019-07-24
 Publication Status: Published in print
 Pages: 17
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Identifiers: DOI: 10.1021/jacs.9b03703
 Degree: -



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Project name : We thank A. Dong (Structural Genomics Consortium, Toronto) for his help with collecting X-ray diffraction data. We also thank Yasir Raouf for assistance in docking studies. This work was supported by the Canadian Institutes of Health Research Training Program in Protein Folding and Interaction Dynamics and an Ontario Graduate Scholarship (T.H.K.), an Ontario Student Opportunity Trust Fund award (P.M.), CREST/JST (A.S.), Natural Sciences and Engineering Research Council of Canada Discovery grants 418679 (R.S.P.) and RGPIN-2015-04877 (E.F.P.), Canadian Institutes of Health Research grant MOP-130461 (R.P.), and the Canada Research Chairs Program (E.F.P.). Molecular dynamics computations were performed on the GPC supercomputer at the SciNet HPC Consortium. SciNet is funded by the Canada Foundation for Innovation under the auspices of Compute Canada, the Government of Ontario, Ontario Research Fund–Research Excellence, and the University of Toronto. Coordinates and structure factors have been deposited in the Protein Data Bank with accession numbers 6QKS, 6QKW, 6QKT, and 6QKU for the apo-form of the Y219F mutant of FAcD, the Y219F-FAc-complex, the Y219F-glycolate complex, and the Y219F-ClAc complex, respectively.
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Source 1

Title: Journal of the American Chemical Society
  Other : J. Am. Chem. Soc.
  Abbreviation : JACS
Source Genre: Journal
Publ. Info: Washington, DC : American Chemical Society
Pages: - Volume / Issue: 141 (29) Sequence Number: - Start / End Page: 11540 - 11556 Identifier: ISSN: 0002-7863
CoNE: https://pure.mpg.de/cone/journals/resource/954925376870