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Statements

Subject Item
n2:RIV%2F61388955%3A_____%2F12%3A00375973%21RIV13-GA0-61388955
rdf:type
skos:Concept n15:Vysledek
dcterms:description
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. 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.
dcterms:title
Role of Glutamate 64 in the Activation of the Prodrug 5-Fluorocytosine by Yeast Cytosine Deaminase Role of Glutamate 64 in the Activation of the Prodrug 5-Fluorocytosine by Yeast Cytosine Deaminase
skos:prefLabel
Role of Glutamate 64 in the Activation of the Prodrug 5-Fluorocytosine by Yeast Cytosine Deaminase Role of Glutamate 64 in the Activation of the Prodrug 5-Fluorocytosine by Yeast Cytosine Deaminase
skos:notation
RIV/61388955:_____/12:00375973!RIV13-GA0-61388955
n15:predkladatel
n16:ico%3A61388955
n4:aktivita
n7:P n7:Z
n4:aktivity
P(GA203/09/1627), P(IAA400400812), P(IAA400400908), Z(AV0Z40400503)
n4:cisloPeriodika
1
n4:dodaniDat
n6:2013
n4:domaciTvurceVysledku
n14:5630134
n4:druhVysledku
n19:J
n4:duvernostUdaju
n12:S
n4:entitaPredkladatele
n5:predkladatel
n4:idSjednocenehoVysledku
166001
n4:idVysledku
RIV/61388955:_____/12:00375973
n4:jazykVysledku
n20:eng
n4:klicovaSlova
transition-state analog; barrier hydrogen-bond; side-chain amides
n4:klicoveSlovo
n8:transition-state%20analog n8:side-chain%20amides n8:barrier%20hydrogen-bond
n4:kodStatuVydavatele
US - Spojené státy americké
n4:kontrolniKodProRIV
[10CD1D2C9BD4]
n4:nazevZdroje
Biochemistry
n4:obor
n18:CF
n4:pocetDomacichTvurcuVysledku
1
n4:pocetTvurcuVysledku
7
n4:projekt
n10:IAA400400812 n10:IAA400400908 n10:GA203%2F09%2F1627
n4:rokUplatneniVysledku
n6:2012
n4:svazekPeriodika
51
n4:tvurceVysledku
Sklenák, Štěpán Wu, Y. Liu, A. Li, Y. Felczak, K. Yan, H. Wang, J.
n4:wos
000298907400050
n4:zamer
n13:AV0Z40400503
s:issn
0006-2960
s:numberOfPages
12
n17:doi
10.1021/bi201540z