Inhibition of an ecto-ATP-diphosphohydrolase by azide

Cell surface ATPases (ecto-ATPases or E-ATPases) hydrolyze extracellular AT P and other nucleotides. Regulation of extracellular nucleotide concentrati on is one of their major proposed functions. Based on enzymatic characteriz ation, the E-ATPases have been divided into two subfamilies, ecto-ATPases a nd ecto-ATP-diphosphohydrolases (ecto-ATPDases). In the presence of either Mg2+ or Ca2+, ecto-ATPDases, including proteins closely related to CD39, hy drolyze nucleoside diphosphates in addition to nucleoside triphosphates and are inhibited by millimolar concentrations of azide, whereas ecto-ATPases appear to lack these two properties. This report presents the first systema tic kinetic study of a purified ecto-ATPDase, the chicken oviduct ecto-ATPD ase (Strobel, R.S., Nagy, A.K., Knowles, A.F, Buegel, J. & Rosenberg, M.O. (1996) J. Biol. Chern. 271, 16323-16331), with respect to ATP and ADP, and azide inhibition. K-m values for ATP obtained at pH 6.4 and 7.4 are 10-30 t imes lower than for ADP and the catalytic efficiency is greater with ATP as the substrate. The enzyme also exhibits complicated behavior toward azide. Variable inhibition by azide is observed depending on nucleotide substrate , divalent ion, and pH. Nearly complete inhibition by 5 mM azide is obtaine d when MgADP is the substrate and when assays are conducted at pH 6-6.4. Az ide inhibition diminishes when ATP is the substrate, Ca2+ as the activating ion, and at higher pH. The greater efficacy of azide in inhibiting ADP hyd rolysis compared to ATP hydrolysis may be related to the different modes of inhibition with the two nucleotide substrates. While azide decreases both V-max and K-m for ADP, it does not alter the K-m for ATP. These results sug gest that the apparent affinity of azide for the E.ADP complex is significa ntly greater than that for the free enzyme or E.ATP. The response of the en zyme to three other inhibitors, fluoride, vanadate, and pyrophosphate, is a lso dependent on substrate and pH. Taken together, these results are indica tive of a discrimination between ADP and ATP by the enzyme. A mechanism of azide inhibition is proposed.