Evidence for Two Different Reactive Sulfhydryl Groups in the ATP‐Binding Sites of (Na⁺+ K⁺)‐ATPase

The disulfide of thioinosine triphosphate, (Sno PPP ) 2 is a substrate of (Na + + K + )‐ATPase from pig kidney. Binding of (Sno PPP ) 2 and of the β,γ‐imido or β,γ‐methylene analogues, (Sno PP [NH] P ) 2 or (Sno PP [CH 2 ] P ) 2 , to (Na + + K + )‐ATPase in the presence of 10 mM EDTA results in an inactivation of the enzyme. The inactivation is temperature‐dependent and has an activation energy of 95.5 kJ/mol with (Sno PPP ) 2 as substrate. The inactivation proceeded with all disulfide analogues of ATP with two different velocities. Identical inactivation rate constants k 2 were found for all substances for the rapidly inactivating part (k′ 2 = 2.9–4.7 × 10 −3 s −1 ) and the slowly inactivating part (k″ 2 = 2.0−2.3 × 10 −4 s −1 ). From the analysis of the inactivation kinetics identical dissociation constants were found for both sites. The dissociation constants of the complexes between enzyme and ATP analogue inactivating the enzyme slowly were: (Sno PPP ) 2 = 18.6 μM, (Sno PP [NH] P ) 2 = 38.8 μM and (Sno PP [CH 2 ] P ) 2 = 63.3 μM. ATP protected both sites against the inactivation by the disulfide analogues of ATP. From the protective action of ATP at 37° C against the inactivation, a dissociation constant of the enzyme‐ATP complex of 2.95 μM was calculated for the rapidly inactivating site and of 77 μM for the slowly reacting site. The total capacity of the sulfhydryl groups reacting with the ATP analogue (Sno PPP ) 2 is four‐times higher than the capacity of the phosphointermediate and the with (Sno PPP ) 2 rapidly reacting site (high, affinity ATP‐binding site). It is concluded that (Sno PPP ) 2 as well as (Sno PP [NH] P ) 2 and (Sto PP [CH 2 ] P ) 2 are ATP affinity labels of the high‐affinity and low‐affinity ATP‐binding sites of (Na + + K + )‐ATPase. Since dithiothreitol reactivates (Na + + K + )‐ATPase after inactivation by (Sno PPP ) 2 [Patzelt‐Wenezler and Schoner (1975) Biochim. Biophys, Acta, 403 , 538–513] it is assumed that (Na + + K + )‐ATPase contains two different reactive types of sulfhydryl groups in the ATP‐binding sites. Tte sulfhydryl group, may interact with the 6‐amino group of the purine moiety of ATP.