Premium
Activation of Two Sequential C‐H bonds in the Thymidylate Synthase‐Catalyzed Reaction
Author(s) -
Islam Zahidul,
Strutzenberg Timothy,
Ghosh Ananda,
Kohen Am
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.572.8
Subject(s) - chemistry , thymidylate synthase , nucleophile , hydride , stereochemistry , methylene , qm/mm , catalysis , cofactor , nucleotide , leaving group , enzyme catalysis , transition state , combinatorial chemistry , enzyme , medicinal chemistry , biochemistry , organic chemistry , hydrogen , medicine , fluorouracil , surgery , chemotherapy , gene
Thymidylate Synthase (TSase) is a crucial enzyme that maintains cellular dTMP (2′‐deoxythymidine‐5′‐monophosphate) pool required for DNA biosynthesis in most living organisms. TSases requires the cofactor methylene tetrahydrofolate (CH 2 H 4 folate) that donates a methylene and subsequently a hydride to the substrate dUMP (2′‐deoxyuridine‐5′‐monophosphate) to form dTMP. Two sequential H‐transfers are at the heart of TSase catalysis: a non rate‐limiting proton abstraction from a carbon and a rate‐limiting hydride transfer between two carbons. QM/MM calculations of these chemical conversions proposed a new mechanism for the TSase catalysis. In the new mechanism, the covalent bond between the enzymatic nucleophile, C146, and the reaction intermediate breaks concertedly with the abstraction of the proton from C5 of the dUMP, leaving an unbound reaction intermediate. QM/MM calculations of a subsequent hydride transfer also predicted a concerted simultaneous C146 elimination from the final product. Key to both calculations was a highly conserved R166 that first activates the C6‐S bond and then stabilizes the transition state for both H‐transfers. We tested the predicted role of R166 in both H‐transfers by comparing the primary KIEs and their temperature dependence for the wild type TSase and its R166K mutant. The temperature dependence of KIEs indicated a crucial contribution of R166 in both C‐H bond activations. It appears that the experimental findings corroborate the predictions of the QM/MM calculations, supporting the existence of a new reaction intermediate as a potential target for the design of antibiotic and chemotherapeutic drugs. This work was supported by NIH R01 GM065368 to AK and Iowa CBB to ZI.(1) Biochemistry 2013 , 52, 2348 (2) J. Am. Chem. Soc. 2011 , 133, 6692.