Ligand-Dependent Conformational Dynamics of Dihydrofolate Reductase

• Type of Material: Article
• Authors: Michael J. Reddish, Emory University; Morgan B. Vaughn, Emory University; Rong Fu, Emory University; R. Brian Dyer, Emory University
• Language: English
• Date: 2016-03-03
• Publisher: American Chemical Society
• Publication Version: Author Accepted Manuscript (After Peer Review)
• Copyright Statement: © 2016 American Chemical Society
• Final Published Version (URL): https://doi.org/10.1021/acs.biochem.5b01364
• Title of Journal or Parent Work: Biochemistry
• Volume: 55
• Issue: 10
• Start Page: 1485
• End Page: 1493
• Grant/Funding Agency: National Institutes of Health; National Science Foundation
• Grant/Funding Information: This work was supported by National Institutes of Health Grant GM068036 (R.B.D.) and by National Science Foundation Graduate Fellowship DGE-0940903 (M.J.R.).
• Keywords: protein–ligand complexes; conformational states; Enzymes; dynamic structural changes; catalysis
• Subject - Topics: Enzymology; Biophysics

Abstract

Enzymes are known to change among several conformational states during turnover. The role of such dynamic structural changes in catalysis is not fully understood. The influence of dynamics in catalysis can be inferred, but not proven, by comparison of equilibrium structures of protein variants and protein–ligand complexes. A more direct way to establish connections between protein dynamics and the catalytic cycle is to probe the kinetics of specific protein motions in comparison to progress along the reaction coordinate. We have examined the enzyme model system dihydrofolate reductase (DHFR) from Escherichia coli with tryptophan fluorescence-probed temperature-jump spectroscopy. We aimed to observe the kinetics of the ligand binding and ligand-induced conformational changes of three DHFR complexes to establish the relationship among these catalytic steps. Surprisingly, in all three complexes, the observed kinetics do not match a simple sequential two-step process. Through analysis of the relationship between ligand concentration and observed rate, we conclude that the observed kinetics correspond to the ligand binding step of the reaction and a noncoupled enzyme conformational change. The kinetics of the conformational change vary with the ligand's identity and presence but do not appear to be directly related to progress along the reaction coordinate. These results emphasize the need for kinetic studies of DHFR with highly specific spectroscopic probes to determine which dynamic events are coupled to the catalytic cycle and which are not.