Abstract
Effects of K+ and Na+ on the transient state kinetics of (Na+ + K+)-ATPase from Electrophorus electricus were examined. Exposure of the enzyme to K+ for brief intervals prior to the addition of ATP and Na+ converts the enzyme to a form (E2 . K) which is transiently less reactive than when all three ligands are added simultaneously. Enzyme is reconverted to the rapidly reacting (E1) form if Na+ is added prior to ATP. Exposure of the ATPase to K+ without Na+ for 1 to 2 h partially restores the initial phosphate (Pi) burst but greatly depresses the amount of phosphoprotein intermediate (E-P) observed. Experiments in the presence of valinomycin suggest that much of this depression in E-P is related to the presence of sealed vesicles with K+ sites sequestered in the interior. Although the results are largely consistent with a simple model in which ATP hydrolysis occurs only through the phosphoenzyme intermediate, the partial restoration of the Pi burst following long term exposure to K+ appears to be attributable to a slow change which may allow some ATP hydrolysis to occur within formation of a phosphoenzyme intermediate.
Highlights
From the Laboratoryof Neurochemistry, National Instituteof Neurological and Communicative Disorders and Stroke, National Institutesof Health, Bethesda, Maryland20205
The results are largely consistent with a simple model in which ATP hydrolysis occurs only through the phosphoenzyme intermediate, the partial restoration of the Pi burst following long term exposure to K’ appears to be attributable to a slow change which may allow some ATP hydrolysis to occur without formation of a phosphoenzyme intermediate
The increase in burst amplitude as [Kf]was raised and various Kf concentrations (0 to 20 mM) with enzyme that was accompanied by a reciprocal decline in the phosphoenhad been premixed with 100mM Naf
Summary
Effects of K’ on Phosphoenzyme Formation a n d P, Liberation-Potassium activation of the (Na’ + K+)-ATPaseis believed to takeplace by activation of the catalytic centerfor hydrolysis of Ez-P (3). When the enzyme was equilibrated with 100 mM Na+ and Effects of Order ofAdditionof Na+ andK+-Equilibration phosphorylated by the addition of 10PM [Y-~'P]ATPplus of the enzyme with both Na+and K+ prior to ATP increased. When the enzymewas equilibrated with Na' andthen mixed with ATP and 20 mM K+, the steady state level of phosphoenzyme decreased while boththe initial and final rates ofPi production increased relative to the observations at 0.5 mM Kf.By extrapolating the steady state phase of Pi production to zero reaction time, the burst amplitude was obtained (Table I). Part or all of the phosphoenzyme may arise from a side reaction of the enzyme that is sensitive to thesequence of ligand additions These hypotheses were tested by examining the transient kinetics of hydrolysis of the phosphoenzyme formed under the experimental conditions of Fig. 2 in a doublemixing experiment. The time course of disappearance of phosphoenzyme followingaddition of excess unlabeled ATP is shown in
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