PHOSPHORYLATION OF THE SODIUM-POTASSIUM ADENOSINE-TRIPHOSPHATASE WITHADENOSINE-TRIPHOSPHATE AND SODIUM-ION THAT REQUIRES SUBCONFORMATIONS IN ADDITION TO PRINCIPAL E(1) AND E(2) CONFORMATIONS OF THE ENZYME

Phosphorylation of the free sodium-potassium adenosinetriphosphatase o f sheep kidney upon the addition of 0.02-2.0 mM ATP and a saturating c oncentration of Na+ (125 mM) follows pseudo-first-order kinetics, The first-order rate constant increases with increasing [ATP] and levels o ff at high [ATP] with a limiting rate constant of 180 s(-1) at 25 degr ees C, pH 7.4, 125 mM NaCl, and 3.0 mM MgCl2, This rate constant is ab out 1/3 Of the maximum rate constant of 460 s(-1) for phosphorylation of enzyme that had been preincubated with Na+ under identical conditio ns [Keillor, J. W., & Jencks, W. P., (1996) Biochemistry 35, 2750-2753 ]; this rate ratio is similar to that for phosphorylation of the calci um ATPase with and without initial incubation with Ca2+ [Petithory, J. R.. & Jencks, W. P. (1986) Biochemistry 25, 4493-4497]. The K-0.5 for ATP is 18 +/- 3 mu M for the free enzyme, which is about 1/4 of K-0.5 = 75 mu M for enzyme that was preincubated with Na+. Addition of ADP to ATP and Na+ decreases the yield of E similar to P progressively wit h increasing ADP concentration. Upon an increase of [ADP] from 0 to 2. 0 mM, the rate constant for phosphorylation decreases 4-fold (167 to 4 1 s(-1)) at low [ATP] (0.25 mM) and about 2.7-fold (174 to 64 s(-1)) a t high [ATP] (2.0 mM). The absence of an induction period for phosphor ylation of E upon the addition of saturating concentrations of ATP and Na+ indicates that all the prior reaction steps are much faster than the rate-limiting step. These results are consistent with a rate-deter mining conformational change of the E . ATP . Na-3 intermediate. The d ecrease of the rate constant with increasing [ADP] is attributed to co mpetition between ATP and ADP for the free enzyme.