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Int. J. Mol. Sci. 2015, 16(4), 7304-7319;

Role of Long-Range Protein Dynamics in Different Thymidylate Synthase Catalyzed Reactions

Department of Chemistry, University of Iowa, Iowa City, IA 52242-1727, USA
Author to whom correspondence should be addressed.
Academic Editors: Tatyana Karabencheva-Christova and Christo Z. Christov
Received: 18 February 2015 / Revised: 26 March 2015 / Accepted: 30 March 2015 / Published: 1 April 2015
(This article belongs to the Collection Proteins and Protein-Ligand Interactions)
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Recent studies of Escherichia coli thymidylate synthase (ecTSase) showed that a highly conserved residue, Y209, that is located 8 Å away from the reaction site, plays a key role in the protein’s dynamics. Those crystallographic studies indicated that Y209W mutant is a structurally identical but dynamically altered relative to the wild type (WT) enzyme, and that its turnover catalytic rate governed by a slow hydride-transfer has been affected. The most challenging test of an examination of a fast chemical conversion that precedes the rate-limiting step has been achieved here. The physical nature of both fast and slow C-H bond activations have been compared between the WT and mutant by means of observed and intrinsic kinetic isotope effects (KIEs) and their temperature dependence. The findings indicate that the proton abstraction step has not been altered as much as the hydride transfer step. Additionally, the comparison indicated that other kinetic steps in the TSase catalyzed reaction were substantially affected, including the order of the substrate binding. Enigmatically, although Y209 is H-bonded to 3'-OH of 2'-deoxyuridine-5'-mono­phosphate (dUMP), its altered dynamics is more pronounced on the binding of the remote cofactor, (6R)-N5,N10-methylene-5,6,7,8-tetrahydrofolate (CH2H4folate), revealing the importance of long-range dynamics of the enzymatic complex and its catalytic function. View Full-Text
Keywords: TSase; KIEs; kinetic isotope effects; WT; wild-type; TRS; tunneling ready state TSase; KIEs; kinetic isotope effects; WT; wild-type; TRS; tunneling ready state

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Abeysinghe, T.; Kohen, A. Role of Long-Range Protein Dynamics in Different Thymidylate Synthase Catalyzed Reactions. Int. J. Mol. Sci. 2015, 16, 7304-7319.

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