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

Luk, Louis Y. P., Ruiz-Pernía, J. Javier, Adesina, Aduragbemi S. ORCID: https://orcid.org/0000-0002-1029-4159, Loveridge, E. Joel, Tuñón, Iñaki, Moliner, Vincent and Allemann, Rudolf K. (2015) Chemical ligation and isotope labeling to locate dynamic effects during catalysis by dihydrofolate reductase. Angewandte Chemie-International Edition, 54 (31). pp. 9016-9020. ISSN 1433-7851

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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 ((2) H, (13) C, (15) N) 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
Additional Information:	© 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
Uncontrolled Keywords:	catalysis,methods,ligation,models, molecular,chemistry
Faculty \ School:	Faculty of Science > School of Biological Sciences
Depositing User:	LivePure Connector
Date Deposited:	24 Jun 2021 00:11
Last Modified:	30 Mar 2024 01:19
URI:	https://ueaeprints.uea.ac.uk/id/eprint/80322
DOI:	10.1002/anie.201503968

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