Volume 213, number 2, 381-384 FEB 4531 March Mode of ~~~i~i~io~ of sodium azide on H%%TPase of E~c~e~~~~~~ coti Takato Noumi, Masatomo Maeda and Masamitsu Futai 1987 zyxwvu Department of Organic Chemistry and Biorhemistry, The institute of ScientijCic and Industrial Research, Osaka I/nrversity, Ibaraki, Osaka 567, Japan Received 20 January 1987 Sodium azide jnh~bit~ n-&i-site ~st~ady-state) AT&se activity of E. cciIi Fr more than 90~~ but did not affect u&site (single-site) ATBase activity. Thus azide inhibited multi-site ATPase activity by fowering cata- lytic cooperativity. Consistent with this observation, azide changed the ligand-induced Ruorescence response ofaurovertin bound to Fr. H+-ATPase; Fr; U&site catalysis; Multi-site catalysis; Sodium azide; fE. C&i) 1. INTRODUCTION The H+-ATPase complex (FIFO) of ~~c~~~~c~~~ co& catalyzes ATP synthesis at the termin& step of oxidative p~~sp~~r~~at~~n (reviews f&-4]). The catalytic portion, Fr, is formed from 5 subunits CY~ fl, y, 6, and f with an Q&Q& stoichiometry and acts as an ATPase. The mechanism of ATP hydrolysis by Fr from mitochondria [5,6] and zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHG E. colt’ [7,X] has been studied extensively: the ATP at the first catalytic site is hydrolyzed only slowly (%mi-site’ hydrolysis), but on binding of ATP at the second and third sites, the ATP at the first site is hydrolyzed with release of products at maximal velocity (‘multi-site’ hydrolysis) due to the positive cooperativity between the three catalytic sites. The ratio of the uni- and multi-site rates is IO5 for mitochondrial Fi and 104-10’ for E. co& Fr. A useful approach in further studies on the catalytic mechanism of Fr is the introduction of Correspondence address: T. Noumi, Dept of Organic Chemistry and Biochemistry, The Institute of Scientific andlndustrial Research, Osaka University, Ibaraki, Osaka 567, Japan Abbreviations: Pi, inorganic phosphate; 32Pi, radioac- tive inorganic phosphate specific inhibitors. Sodium azide is known to be a potent inhibitor of Fr-ATPase [9,10], although its mode of inhibition has nut been studied in detail. Recently we found that FX from a mutant strain RF43 (defective in the ~-subunit) has a residual multi-site ATPase activity that is insensitive to azide f8J. Senior and co-workers fll,l2] showed that the residual multi-site activities of Fr from uncA mutants (defective in the cr-subunit) and revertants of uncAlO2 were also insensitive to azide, The Frs from strains KF43 and ~~4402 both had similar uni-site ATPase activity to wild- type Fr, although their multi-site activities were l-3% of that of wild type. These findings suggest that the interaction between the CY- and ~~subunits is essentia’f for the positive coo~rativity and may be responsible for the azide sensitivity. In this work we examined the effects of azide on the kinetics of uni- and multi-site ATP hydrolyses and on the conformational changes of Fr on its bin- dings to ADP, ATP and Mgzc. 2. MATERIALS AND METHODS Fr was prepared from E. coli strain MLJOS-225 as described 1131. The specific activity of the preparation (multi-site or steady-state activity) was