Thymidylate Synthase (TSase), a key enzyme for the DNA biosynthetic pathway, catalyzes the conversion of deoxyuridylate (dUMP) to thymidylate (dTMP). TSase is a chemotherapeutic drug target, as blocking of this enzyme causes thymine-less cell death. The cofactor methylene tetrahydrofolate (CH2H4folate) first transfers a methylene group and subsequently a hydride, and thus a net methyl group substitutes for the C5 proton of dUMP to form dTMP. TSase is a highly conserved enzyme, and TSases from pathogenic organisms exhibit high sequence identity with its human counterpart (hsTSase) [1]. Microbial TSases have been utilized as models to explore the mechanism and for drug designs. However, some unique characteristics of hsTSase such as presence of an alternative inactive conformation [2] and no rate enhancement by Mg2+ [3] indicate that the kinetic and mechanistic features of mammalian TSases differ from the one present in microbial sources. A large number of structural, kinetic and mechanistic information are available for the TSase from pathogenic organisms whereas a handful of those features are known for hsTSase. Extensive investigations of mammalian TSases might uncover unique properties of hsTSase that could be exploited for constructing antimicrobial as well as finely-tuned anticancer drugs. Here we aim to explore kinetic and H-transfer features of hsTSase. While the Michaelis-Menten kinetic parameters for hsTSase seem to be similar to that for E. coli TSase (ecTSase), the substrates binding mechanism for the former appears to be less ordered than for the later. Activation of the C5 proton of dUMP was investigated by the temperature dependence of intrinsic KIEs, which indicates that the proton transfer in hsTSase might have a slightly different transition state structure than the ecTSase. Investigation of the activation of the hydride transfer from the CH2H4folate is underway, which could identify more interesting and distinguishing characteristics of the human TSases. References (1) Carosati, E.; Tochowicz, A.; Marverti, G.; Guaitoli, G.; Benedetti, P.; Ferrari, S.; Stroud, R. M.; Finer-Moore, J.; Luciani, R.; Farina, D.; Cruciani, G.; Costi, M. P. J. Med. Chem. 2012, 55, 10272-10276. (2) Gibson, L. M.; Lovelace, L. L.; Lebioda, L. Biochemistry 2008, 47, 4636-4643. (3) Wang, Z.; Sapienza, P. J.; Abeysinghe, T.; Luzum, C.; Lee, A. L.; Finer-Moore, J. S.; Stroud, R. M.; Kohen, A. Journal of the American Chemical Society 2013, 135, 7583-7592.

H-transfer mechanisms in human vs E. coli thymidylate synthase

251st National Meeting (2016)

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