Abstract

Thymidine triphosphate monophosphohydrolase (dTTPase), an enzyme which catalyzes the hydrolysis of dTTP to the corresponding diphosphate (dTDP), has been purified to homogeneity from human serum. The enzyme sediments with 3.8 S in sucrose density gradients. A Stokes radius of 31 A is estimated by gel filtration. Accordingly, its molecular weight is 48 500. Since only one single band of Mr 24 000 is detected after sodium dodecyl sulfate/polyacrylamide gel electrophoresis, the native enzyme seems to be composed of two identical subunits. The enzyme exhibits optimal activity over a pH range from 7 to 9, and the activation energy is estimated to be 7.1 kcal/mol (29.7 kJ/mol) at pH 7.8. While the enzyme is active in the absence of added divalent cations, the activity can be inhibited by ethylenediaminetetracetic acid (EDTA) but not by phenanthroline. The inhibition caused by EDTA is reversed by Mn2+. Zn2+ causes a complete inhibition of enzyme activity. No requirement exists for a sulfhydryl compound. The enzyme has an Rf value of 0.45, an isoelectric point of 5.2, and an apparent Km value of 40 microM for dTTP. dUTP and UTP are degraded by about 50 and 20% of the rate of dTTP hydrolysis, respectively. Other deoxyribonucleosides or ribonucleoside triphosphates do not serve as substrates for the dTTPase. The existence of this enzyme is significant since it could play a role in the regulation of the cellular dTTP levels.