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Chemical ligation and isotope labeling to locate dynamic effects during catalysis by dihydrofolate reductase

Luk, Louis Yu Pan, Ruiz-Pernía, J. Javier, Adesina, Aduragbemi Sunday, Loveridge, Edric Joel, Tuñón, Iñaki, Moliner, Vincent and Allemann, Rudolf Konrad 2015. Chemical ligation and isotope labeling to locate dynamic effects during catalysis by dihydrofolate reductase. Angewandte Chemie - International Edition 54 (31) , pp. 9016-9020. 10.1002/anie.201503968

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Abstract

Chemical ligation has been used to alter motions in specific regions of dihydrofolate reductase from E. coli and to investigate the effects of localized motional changes on enzyme catalysis. Two isotopic hybrids were prepared; one with the mobile N-terminal segment containing heavy isotopes (2H, 13C, 15N) and the remainder of the protein with natural isotopic abundance, and the other one with only the C-terminal segment isotopically labeled. Kinetic investigations indicated that isotopic substitution of the N-terminal segment affected only a physical step of catalysis, whereas the enzyme chemistry was affected by protein motions from the C-terminal segment. QM/MM studies support the idea that dynamic effects on catalysis mostly originate from the C-terminal segment. The use of isotope hybrids provides insights into the microscopic mechanism of dynamic coupling, which is difficult to obtain with other studies, and helps define the dynamic networks of intramolecular interactions central to enzyme catalysis.

Item Type: Article
Date Type: Publication
Status: Published
Schools: Chemistry
Subjects: Q Science > QD Chemistry
Uncontrolled Keywords: chemical ligation; enzyme catalysis; isotope effects; microscopic mechanisms; protein dynamics
Publisher: Wiley-Blackwell
ISSN: 1433-7851
Funders: BBSRC
Date of First Compliant Deposit: 30 March 2016
Date of Acceptance: 22 May 2015
Last Modified: 05 Jun 2017 05:02
URI: http://orca.cf.ac.uk/id/eprint/75394

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