Phosphorylation of the sodium-potassium adenosinetriphosphatase with adenosine triphosphate and sodium ion that requires subconformations in addition to principal E1 and E2 conformations of the enzyme.
Phosphorylation of the free sodium-potassium adenosinetriphosphatase of sheep kidney upon the addition of 0.02-2.0 mM ATP and a saturating concentration of Na+ (125 mM) follows pseudo-first-order kinetics. The first-order rate constant increases with increasing [ATP] and levels off at high [ATP] with a limiting rate constant of 180 s-1 at 25 degrees C, pH 7.4, 125 mM NaCl, and 3.0 mM MgCl2. This rate constant is about 1/3 of the maximum rate constant of 460 s-1 for phosphorylation of enzyme that had been preincubated with Na+ under identical conditions [Keillor, J. W., & Jencks, W. P., (1996) Biochemistry 35, 2750-2753]; this rate ratio is similar to that for phosphorylation of the calcium ATPase with and without initial incubation with Ca2+ [Petithory, J. R., & Jencks, W. P. (1986) Biochemistry 25, 4493-4497]. The K0.5 for ATP is 18 +/- 3 microM for the free enzyme, which is about 1/4 of K0.5 = 75 microM for enzyme that was preincubated with Na+. Addition of ADP to ATP and Na+ decreases the yield of E approximately P progressively with increasing ADP concentration. Upon an increase of [ADP] from 0 to 2.0 mM, the rate constant for phosphorylation decreases 4-fold (167 to 41 s-1) at low [ATP] (0.25 mM) and about 2.7-fold (174 to 64 s-1) at high [ATP] (2.0 mM). The absence of an induction period for phosphorylation 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-determining conformational change of the E.ATP.Na3 intermediate. The decrease of the rate constant with increasing [ADP] is attributed to competition between ATP and ADP for the free enzyme.
Ghosh
M C
MC
Graduate Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02254-9110, USA.
Jencks
W P
WP
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D008956:Models, Chemical; D010766:Phosphorylation; D011487:Protein Conformation; D012756:Sheep; D012964:Sodium / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000378:metabolism*
D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase