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Role of Glutamate 64 in the Activation of the Prodrug 5-Fluorocytosine by Yeast Cytosine Deaminase
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SYSNO ASEP 0375973 Druh ASEP J - Článek v odborném periodiku Zařazení RIV J - Článek v odborném periodiku Poddruh J Článek ve WOS Název Role of Glutamate 64 in the Activation of the Prodrug 5-Fluorocytosine by Yeast Cytosine Deaminase Tvůrce(i) Wang, J. (US)
Sklenák, Štěpán (UFCH-W) RID, ORCID, SAI
Liu, A. (US)
Felczak, K. (US)
Wu, Y. (US)
Li, Y. (US)
Yan, H. (US)Zdroj.dok. Biochemistry. - : American Chemical Society - ISSN 0006-2960
Roč. 51, č. 1 (2012), s. 475-486Poč.str. 12 s. Jazyk dok. eng - angličtina Země vyd. US - Spojené státy americké Klíč. slova transition-state analog ; barrier hydrogen-bond ; side-chain amides Vědní obor RIV CF - Fyzikální chemie a teoretická chemie CEP IAA400400812 GA AV ČR - Akademie věd IAA400400908 GA AV ČR - Akademie věd GA203/09/1627 GA ČR - Grantová agentura ČR CEZ AV0Z40400503 - UFCH-W (2005-2011) UT WOS 000298907400050 DOI 10.1021/bi201540z Anotace Yeast cytosine deaminase (yCD) catalyzes the hydrolytic deamination of cytosine to uracil as well as the deamination of the prodrug 5-fluorocytosine (5FC) to the anticancer drug 5-fluorouracil. In this study, the role of Glu64 in the activation of the prodrug 5FC was investigated by site-directed mutagenesis, biochemical, nuclear magnetic resonance (NMR), and computational studies. Steady-state kinetics studies showed that the mutation of Glu64 causes a dramatic decrease in k(cat) and a dramatic increase in K, indicating Glu64 is important for both binding and catalysis in the activation of 5FC. (19)F NMR experiments showed that binding of the inhibitor 5-fluoro-1H-pyrimidin-2-one (5FPy) to the wild-type yCD causes an upfield shift, indicating that the bound inhibitor is in the hydrated form, mimicking the transition state or the tetrahedral intermediate in the activation of 5FC. However, binding of 5FPy to the E64A mutant enzyme causes a downfield shift, indicating that the bound 5FPy remains in an unhydrated form in the complex with the mutant enzyme. (1)H and (15)N NMR analysis revealed trans-hydrogen bond D/H isotope effects on the hydrogen of the amide of G1u64, indicating that the carboxylate of Glu64 forms two hydrogen bonds with the hydrated 5FPy. ONIOM calculations showed that the wild-type yCD complex with the hydrated form of the inhibitor 1H-pyrimidin-2-one is more stable than the initial binding complex, and in contrast, with the E64A mutant enzyme, the hydrated inhibitor is no longer favored and the conversion has a higher activation energy, as well. The hydrated inhibitor is stabilized in the wild-type yCD by two hydrogen bonds between it and the carboxylate of Glu64 as revealed by (1)H and (15)H NMR analysis. To explore the functional role of Glu64 in catalysis, we investigated the deamination of cytosine catalyzed by the E64A mutant by ONIOM calculations. Pracoviště Ústav fyzikální chemie J.Heyrovského Kontakt Michaela Knapová, michaela.knapova@jh-inst.cas.cz, Tel.: 266 053 196 Rok sběru 2013
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