Effect of an Asp80Ala substitution on the binding of dUTP and dUMP to Trypanosoma cruzi dUTPase

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• 作者：Ramiro Té ; llez-Sanz ; Zeyad Yassin ; Victor Bernier-Villamor ; Emilia Ortiz-Salmeró ; n ; Juan Alex ; er Musso-Buendia ; Carmen Baró ; n ; Luis Miguel Ruí ; z-Pé ; rez ; Dolores Gonzá ; lez-Pacan
• 关键词：Deoxyuridine 5′ ; -triphosphate nucleotide hydrolase ; Trypanosoma cruzi ; Deoxyuridine 5′ ; -monophosphate ; Deoxyuridine 5′ ; -triphosphate ; Microcalorimetry ; Fluorescence
• 刊名：Biochimie
• 出版年：2007
• 期刊代码：24_03009084
• 类别：bio
• 出版时间：August 2007
• 卷：89
• 期：8
• 页码：972-980
• 文件大小：454 K

摘要

dUTPase (deoxyuridine 5′-triphosphate nucleotide hydrolase) is an enzyme responsible for maintaining low levels of intracellular dUTP and thus prevents uracil incorporation into DNA by DNA polymerases during replication and repair processes. The thermodynamics of binding for both dUTP and dUMP (deoxyuridine 5′-monophosphate) to the D80A mutant form of Trypanosoma cruzi dUTPase have been investigated by fluorescence spectroscopy and high-sensitivity isothermal titration calorimetry. In the presence of magnesium, approximately a 30-fold decrease in the value of the k_cat and a 15-fold increase in the K_m for dUTP hydrolysis was calculated while a 5-fold decrease was observed in the affinity for dUMP. In the absence of magnesium, the affinity for dUTP binding was similar for both enzymes while that for dUMP was lowered 3-fold as a consequence of the mutation. Calorimetric titrations in several buffers with different ionization heats rendered similar proton exchanges during the binding of dUMP. Thus, apparently the side chain of Asp 80 does not seem to vary its protonation state during the binding process. The enthalpy change values for the D80A mutant hardly change with temperature and, in addition, were Mgp>2+p> independent. We conclude that the D80A mutation induces only a slight conformational change in the active site yet results in a significant alteration of nucleotide binding and modifies the ability of the enzyme to discriminate between dUTP and dUMP when magnesium is present.