title: Conformational change of sodium- and potassium-dependent adenosine triphosphatase. Conformational evidence for the Post-Albers mechanism in Na+- and K+-dependent hydrolysis of ATP.
abstract: The addition of ATP with K+ to pig kidney Na+,K+-ATPase (EC 3.6.1.3) modified with a sulfhydryl fluorescent reagent N-[p-(2-benzimidazolyl)phenyl]maleimide induced a transient decrease (t 1/2 = 0.01 s) in the fluorescence in the presence of Mg2+ with 0.64 M Na+, followed by a slow increase (t 1/2 = 0.08 s), to give a higher steady level than that observed without K+. The addition induced a transient increase (t 1/2 less than 0.02 s) in the amount of phosphoenzyme, followed by a slow decrease (t 1/2 = 0.08 s), but the addition without K+ induced a monophasic increase (t 1/2 = 0.02 s). The addition of ATP in the presence of 2 M Na+ with Ca2+ induced a monophasic decrease (t 1/2 = 0.1 s) in the fluorescence along with a much slower increase (t 1/2 = 1.2 s) in the amount of phosphoenzyme. No significant burst of acid-labile phosphate was observed. The data showed clearly the accumulation of the enzyme-ATP complex preceding the phosphoenzyme formation. Fluorescence intensity of these enzyme species and the amount of phosphoenzyme permitted the simulation using the reaction mechanism including enzyme-ATP complex, ADP-sensitive phosphoenzyme, K+-sensitive phosphoenzyme, and K+-bound enzyme. The simulation gave a good fit to the experimental data which showed that ATP is hydrolyzed in sequence through the above intermediates in the presence of both Na+ and K+.
mesh_terms: D000818:Animals; D006868:Hydrolysis; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D008301:Maleimides / Q000494:pharmacology; D008954:Models, Biological; D011487:Protein Conformation; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013552:Swine
chemical_list: D008301:Maleimides; D013439:Sulfhydryl Reagents; C000245:N-(4-(2-benzimidazolyl)phenyl)maleimide; D000254:Sodium-Potassium-Exchanging ATPase