Open AccessInhibition of an ecto‐ATP‐diphosphohydrolase by azide
Author(s) -
Knowles Aileen F.,
Nagy Agnes K.
Publication year - 1999
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.1432-1327.1999.00389.x
Subject(s) - azide , nucleotide , atp hydrolysis , atpase , sodium azide , nucleoside , chemistry , nucleoside triphosphate , substrate (aquarium) , extracellular , divalent , biochemistry , enzyme , stereochemistry , biophysics , biology , organic chemistry , ecology , gene
Cell surface ATPases (ecto‐ATPases or E‐ATPases) hydrolyze extracellular ATP and other nucleotides. Regulation of extracellular nucleotide concentration is one of their major proposed functions. Based on enzymatic characterization, the E‐ATPases have been divided into two subfamilies, ecto‐ATPases and ecto‐ATP‐diphosphohydrolases (ecto‐ATPDases). In the presence of either Mg 2+ or Ca 2+ , ecto‐ATPDases, including proteins closely related to CD39, hydrolyze 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 systematic kinetic study of a purified ecto‐ATPDase, the chicken oviduct ecto‐ATPDase (Strobel, R.S., Nagy, A.K., Knowles, A.F., Buegel, J. & Rosenberg, M.O. (1996) J. Biol. Chem. 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 times 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 m m azide is obtained when MgADP is the substrate and when assays are conducted at pH 6–6.4. Azide inhibition diminishes when ATP is the substrate, Ca 2+ as the activating ion, and at higher pH. The greater efficacy of azide in inhibiting ADP hydrolysis 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 suggest that the apparent affinity of azide for the E·ADP complex is significantly greater than that for the free enzyme or E·ATP. The response of the enzyme to three other inhibitors, fluoride, vanadate, and pyrophosphate, is also dependent on substrate and pH. Taken together, these results are indicative of a discrimination between ADP and ATP by the enzyme. A mechanism of azide inhibition is proposed.