Kinetic properties and inhibition of the dimeric dUTPase-dUDPase from Leishmania major.
Kinetic properties of the dimeric enzyme dUTPase from Leishmania major were studied using a continuous spectrophotometric method. dUTP was the natural substrate and dUMP and PPi the products of the hydrolysis. The trypanosomatid enzyme exhibited a low K(m) value for dUTP (2.11 microM), a k(cat) of 49 s(-1), strict Michaelis-Menten kinetics and is a potent catalyst of dUDP hydrolysis, whereas in other dUTPases described, this compound acts as a competitive inhibitor. Discrimination is achieved for the base and sugar moiety showing specificity constants for different dNTPs similar to those of bacterial, viral, and human enzymes. In the alkaline range, the K(m) for dUTP increases with the dissociation of ionizable groups showing pK(a) values of 8.8, identified as the uracil moiety of dUTP and 10, whereas in the acidic range, K(m) is regulated by an enzyme residue exhibiting a pK(a) of 7.1. Activity is strongly inhibited by the nucleoside triphosphate analog alpha-beta-imido-dUTP, indicating that the enzyme can bind triphosphate analogs. The existence of specific inhibition and the apparent structural and kinetic differences (reflected in different binding strength of dNTPs) with other eukaryotic dUTPases suggest that the present enzyme might be exploited as a target for new drugs against leishmaniasis.
Hidalgo-Zarco
F
F
Instituto de Parasitología y Biomedicina López-Neyra, 18001-Granada, Spain.
Camacho
A G
AG
Bernier-Villamor
V
V
Nord
J
J
Ruiz-Pérez
L M
LM
González-Pacanowska
D
D
D000818:Animals; D002412:Cations / Q000494:pharmacology; D003856:Deoxyuracil Nucleotides / Q000378:metabolism; D019281:Dimerization; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D018320:Leishmania major / Q000201:enzymology*; D011755:Pyrophosphatases / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature
D002412:Cations; D003856:Deoxyuracil Nucleotides; C027078:deoxyuridine triphosphate; D011755:Pyrophosphatases; C020020:dUTP pyrophosphatase