A potent nucleoside triphosphate hydrolase from the parasitic protozoan Toxoplasma gondii. Purification, some properties, and activation by thiol compounds.
A potent nucleoside triphosphate hydrolase (EC 3.6.1.3) with a number of unusual properties has been found in the parasitic protozoan (Toxoplasma gondii) and has been purified to homogeneity. The enzyme is localized in the cytosol and constitutes 3-4% of the total cytosolic protein. It has a molecular weight of 240,000-260,000 and contains four equivalent subunits of Mr = 63,000. Dithiol compounds such as dithiothreitol, dithioerythritol, or dimercaptopropanol were essential activators of the enzyme. Monothiol compounds had no effect. The specific activity of the purified enzyme was 2,500 mumol/min/mg at 37 degrees C under optimal conditions. Magnesium was the most effective activating metal ion, although manganese and calcium were also active. A higher excess of magnesium over total ATP was essential for maximal activity. Anions were found to inhibit the enzyme activity in an almost chaotropic order. The enzyme demonstrated a wide substrate specificity for both ribo- and deoxyribonucleoside triphosphate and hydrolyzed these nucleotides at almost the same rate. ADP was also a substrate and was hydrolyzed at a rate of 18% of that for ATP. Slight activity was seen with inorganic tri- and tetrapolyphosphates but not with monophosphate compounds. Km values for MgATP2- and MgADP- were 0.12 +/- 0.01 mM and 0.70 +/- 0.06 mM, respectively.
D000818:Animals; D003600:Cytosol / Q000201:enzymology; D004789:Enzyme Activation; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D043583:Nucleoside-Triphosphatase; D010744:Phosphoric Monoester Hydrolases / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000494:pharmacology*; D014122:Toxoplasma / Q000201:enzymology*
D046911:Macromolecular Substances; D013438:Sulfhydryl Compounds; D010744:Phosphoric Monoester Hydrolases; D043583:Nucleoside-Triphosphatase