2024-03-29T14:26:35Zhttps://repositori.uji.es/oai/requestoai:repositori.uji.es:10234/956932022-02-02T18:27:54Zcom_10234_7013com_10234_9col_10234_8638
00925njm 22002777a 4500
dc
Kanaan Izquierdo, Natalia
author
Martí Forés, Sergio
author
Moliner, Vicent
author
Kohen, Amnon
author
2009
Thymidylate synthase (TS) is an enzyme that catalyzes a complex cascade of reactions. A theoretical study of the reduction of an exocyclic methylene intermediate by hydride transfer from the 6S position of 5,6,7,8-tetrahydrofolate (H4folate), to form 2′-deoxyuridine 5′-monophosphate (dTMP) and 7,8-dihydrofolate (H2folate), has been carried out using hybrid quantum mechanics/molecular mechanics methods. This step is of special interest because it is the rate-limiting step of the reaction catalyzed by TS. The acceptor of this hydride is an intermediate that is covalently bound to the enzyme via a thioether bond to an overall conserved active site cysteine residue (Cys146 in Escherichia coli). Heretofore, whether the hydride transfer precedes the thiol abstraction that releases the product from the enzyme or whether these two processes are concerted has been an open question. We have examined this step in terms of free energy surfaces obtained at the same temperatures we previously used in experimental studies of this mechanistic step (273−313 K). Analysis of the results reveals that substantial features of the reaction and the nature of the H-transfer seem to be temperature independent, in agreement with our experimental data. The findings also indicate that the hydride transfer and the scission of Cys146 take place in a concerted but asynchronous fashion. This 1,3-SN2 substitution is assisted by arginine 166 and several other arginine residues in the active site that polarize the carbon−sulfur bond and stabilize the charge transferred from cofactor to substrate. Finally, the simulation elucidates the molecular details of the enzyme’s motion that brings the system to its transition state and, in accordance with the experimental data, indicates that this “tunneling ready” conformation is temperature independent.
1089-5639
http://hdl.handle.net/10234/95693
http://dx.doi.org/ 10.1021/jp810548d
thymidylate synthase
Enzymes
QM/MM study of thymidylate synthase: enzymatic motions and the temperature dependence of the rate limiting step