Phosphorylation of the sodium--potassium adenosinetriphosphatase proceeds through a rate-limiting conformational change followed by rapid phosphoryl transfer

Biochemistry
J W Keillor, W P Jencks

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.

References

Jan 1, 1990·Annual Review of Biochemistry·I M Glynn, S J Karlish
Jun 8, 1993·Biochimica Et Biophysica Acta·J D Robinson, P R Pratap
Jan 1, 1967·Annual Review of Biochemistry·R W Albers

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Citations

Apr 24, 2009·Journal of Biomolecular Structure & Dynamics·Rithvik Vinekar, Indira Ghosh
Aug 10, 2004·Biophysical Journal·R Daniel Peluffo
Sep 22, 2001·Biophysical Journal·C LüpfertR J Clarke
Nov 14, 2008·Biochemistry·Anne Pilotelle-BunnerRonald J Clarke
Jun 5, 2021·Biochemical and Biophysical Research Communications·Paula GrenonHugo P Adamo

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