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Chemical Ligation and Isotope Labeling to Locate Dynamic Effects during Catalysis by Dihydrofolate Reductase
dc.contributor.author | Luk, Louis Y. P. | |
dc.contributor.author | Ruiz-Pernía, José Javier | |
dc.contributor.author | Adesina, Aduragbemi S. | |
dc.contributor.author | Loveridge, E. Joel | |
dc.contributor.author | Tuñón, Iñaki | |
dc.contributor.author | Moliner, Vicent | |
dc.contributor.author | Allemann, Rudolf K. | |
dc.date.accessioned | 2016-05-02T08:02:23Z | |
dc.date.available | 2016-05-02T08:02:23Z | |
dc.date.issued | 2015-07-27 | |
dc.identifier.citation | LUK, Louis Y. P.; RUIZ PERNÍA, J. Javier; ADESINA, Aduragbemi S.; LOVERIDGE, E. Joel; TUÑÓN, Iñaki; MOLINER IBÁÑEZ, Vicent; ALLEMANN, Rudolk K..Chemical Ligation and Isotope Labeling to Locate Dynamic Effects during Catalysis by Dihydrofolate Reductase. Angewandte Chemie (2015), v. 54, issue 31, pp. 9016-9020 | ca_CA |
dc.identifier.uri | http://hdl.handle.net/10234/159084 | |
dc.description.abstract | Experimental and computational approaches have long been employed to define the role of protein motions in en- zyme catalysis, but a refined experimental method for locating the origin of dynamic effects has not previously been developed. Here a general synthetic approach is described to alter motions in specific regions of an enzyme and to analyze the effects of such localized motional changes by measuring ‘segment kinetic isotope effects’. Two isotopic hybrids of dihydrofolate reductase from Escherichia coli (EcDHFR) were prepared by chemical ligation; one with the mobile N-terminal segment (amino acids 1-28) con- taining heavy isotopes (2H, 13C, 15N) and the remainder of the protein (amino acids 29-159) with natural isotopic abundance, and the complementary hybrid with the C-terminal segment isotopically labeled. Investigation of the catalytic properties of these hybrids indicated that isotopic substitution of the N-terminal segment affects a physical step of catalysis, whereas the hydride transfer itself is affected by dynamic effects originating from residues 29-159. To verify the effectiveness of this method and to decipher its mechanistic basis, the experimental results were complemented with QM/MM computational studies, rendering a good agreement with experiments and supporting the idea of small dynamic effects on catalysis originated on the C-terminal segment. which indi- cated that segment isotope labeling affects the recrossing trajectories on the reaction barrier. By isolating the effect of the motions of individual regions of the enzyme on the observed rate constants, segment kinetic isotope effects provide insight difficult to ob- tain with other experimental methods. Measurements of segment kinetic isotope effects will help to define the dynamic networks of intramolecular interactions central to enzyme catalysis. | ca_CA |
dc.description.sponsorShip | This work was supported by Cardiff University, grants BB/J005266/1 and BB/L020394/1 (R.K.A.) from the UK’s Bio- technology and Biological Sciences Research Council (BBSRC), by FEDER and Ministerio de Economía y Competitividad funds (project CTQ2012-36253-C03), Generalitat Valenciana (ACOMP/2014/277 and PrometeoII/2014/022) and by Universitat Jaume I (Project P1%1B2011-23) | ca_CA |
dc.format.extent | 7 p. | ca_CA |
dc.format.mimetype | application/pdf | ca_CA |
dc.language.iso | eng | ca_CA |
dc.publisher | Wiley | ca_CA |
dc.relation.isPartOf | Angewandte Chemie (2015), v. 54, issue 31 | ca_CA |
dc.rights.uri | http://rightsstatements.org/vocab/CNE/1.0/ | * |
dc.subject | Chemical ligation | ca_CA |
dc.subject | Dihydrofolate reductase | ca_CA |
dc.subject | E. coli | ca_CA |
dc.subject | Kinetic investigations | ca_CA |
dc.subject | Proteins | ca_CA |
dc.subject | Isotope hybrids | ca_CA |
dc.title | Chemical Ligation and Isotope Labeling to Locate Dynamic Effects during Catalysis by Dihydrofolate Reductase | ca_CA |
dc.type | info:eu-repo/semantics/article | ca_CA |
dc.identifier.doi | http://dx.doi.org/10.1002/anie.201503968 | |
dc.rights.accessRights | info:eu-repo/semantics/openAccess | ca_CA |
dc.relation.publisherVersion | http://onlinelibrary.wiley.com/doi/10.1002/anie.v54.31/issuetoc | ca_CA |
dc.type.version | info:eu-repo/semantics/acceptedVersion | ca_CA |
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