title: Role of long-range protein dynamics in different thymidylate synthase catalyzed reactions.
abstract: 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'-mono-phosphate (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.
authors.affiliation: Department of Chemistry, University of Iowa, Iowa City, IA 52242-1727, USA. donthelma-abeysinghe@uiowa.edu.
authors.forename: Thelma
authors.initials: T
authors.lastname: Abeysinghe
authors.affiliation: Department of Chemistry, University of Iowa, Iowa City, IA 52242-1727, USA. amnon-kohen@uiowa.edu.
authors.forename: Amnon
authors.initials: A
authors.lastname: Kohen
mesh_terms: D002384:Catalysis; D003856:Deoxyuracil Nucleotides / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D007700:Kinetics; D011506:Proteins / Q000737:chemistry*; D011522:Protons; D013696:Temperature; D013763:Tetrahydrofolates / Q000737:chemistry; D013816:Thermodynamics; D013940:Thymidylate Synthase / Q000737:chemistry*
chemical_list: D003856:Deoxyuracil Nucleotides; D011506:Proteins; D011522:Protons; D013763:Tetrahydrofolates; C013123:5,10-methylenetetrahydrofolic acid; C030371:5,6,7,8-tetrahydrofolic acid; C007267:2'-deoxyuridylic acid; D013940:Thymidylate Synthase
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