Effect of an Asp80Ala substitution on the binding of dUTP and dUMP to Trypanosoma cruzi dUTPase.
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 Mg(2+) 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.
Téllez-Sanz
Ramiro
R
Departamento de Química Física, Bioquímica y Química Inorgánica, Facultad de Ciencias Experimentales, Universidad de Almería, La Cañada de San Urbano, 04120 Almería, Spain.
Yassin
Zeyad
Z
Bernier-Villamor
Victor
V
Ortiz-Salmerón
Emilia
E
Musso-Buendia
Juan Alexander
JA
Barón
Carmen
C
Ruíz-Pérez
Luis Miguel
LM
González-Pacanowska
Dolores
D
García-Fuentes
Luis
L
D000409:Alanine / Q000235:genetics / Q000378:metabolism; D019943:Amino Acid Substitution; D000818:Animals; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D002151:Calorimetry; D003856:Deoxyuracil Nucleotides / Q000378:metabolism*; D019281:Dimerization; D007700:Kinetics; D015636:Magnesium Chloride / Q000378:metabolism; D011755:Pyrophosphatases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013696:Temperature; D013816:Thermodynamics; D014349:Trypanosoma cruzi / Q000201:enzymology*
D003856:Deoxyuracil Nucleotides; D015636:Magnesium Chloride; C027078:deoxyuridine triphosphate; D001224:Aspartic Acid; C007267:2'-deoxyuridylic acid; D011755:Pyrophosphatases; C020020:dUTP pyrophosphatase; D000409:Alanine