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 trypanosoma tid enzyme exhibited a low K-m value for dUTP (2.11 muM), a k(cat) of 49 s( -1), strict Michaelis-Menten kinetics and is a potent catalyst of dUDP hydr olysis, whereas in other dUTPases described, this compound acts as a compet itive inhibitor. Discrimination is achieved for the base and sugar moiety s howing specificity constants for different dNTPs similar to those of bacter ial, viral, and human enzymes. In the alkaline range, the K-m for dUTP incr eases 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 ran ge, K-m is regulated by an enzyme residue exhibiting a pK(a) of 7.1. Activi ty is strongly inhibited by the nucleoside triphosphate analog alpha-beta - imido-dUTP, indicating that the enzyme can bind triphosphate analogs. The e xistence of specific inhibition and the apparent structural and kinetic dif ferences (reflected in different binding strength of dNTPs) with other euka ryotic dUTPases suggest that the present enzyme might be exploited as a tar get for new drugs against leishmaniasis.