title: Phosphorylation of the sodium--potassium adenosinetriphosphatase proceeds through a rate-limiting conformational change followed by rapid phosphoryl transfer.
abstract: The sodium-potassium adenosinetriphosphatase of sheep kidney, preincubated with sodium and magnesium (E.Nae), reacts with 0.01-2.00 mM ATP to form covalent phosphoenzyme (E-P). The first order rate constant for phosphorylation increases hyperbolically with ATP concentration with a maximum value of (4.6 +/- 0.9) x 10(2) s-1 and K0.5 = 75 +/- 25 microM (ph 7.4, 25 degrees C, 120 mM NaCl, and 3 mM MgCl2). If the phosphoryl-transfer step were rate-limiting, the approach to equilibrium to give 50% E-P in the presence of ADP would follow kobsd=Kf+Kr+9.2 x 10(2) s-1. However, the formation of phosphoenzyme from E.Na3 with 1.0 mM ATP plus 2.0 mM ADP proceeds to 50% completion with kobsd=(4.2 +/- 0.8) x 10(2) s-1. This result show that phosphoryl transfer from bound ATP to the enzyme is not the rate limiting step for phosphoenzyme formation from E.Na3. The result is consistent with a rate-limiting conformational change of the E.Na3.ATP intermediate that is followed by rapid phosphoryl transfer, with kcat > or = 3000 s-1.
authors.affiliation: Graduate Department of Biochemistry, Brandeis University, Waltham Massachusetts 02254-9110, USA.
authors.forename: J W
authors.initials: JW
authors.lastname: Keillor
authors.forename: W P
authors.initials: WP
authors.lastname: Jencks
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D066298:In Vitro Techniques; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D008956:Models, Chemical; D010766:Phosphorylation; D011487:Protein Conformation; D012756:Sheep; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000254:Sodium-Potassium-Exchanging ATPase