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Studies of the phosphoenzyme intermediate of the yeast plasma membrane proton-translocating ATPase.

Publication ,  Journal Article
Smith, KE; Hammes, GG
Published in: J Biol Chem
September 25, 1988

The yeast plasma membrane proton-pumping ATPase forms a phosphorylated intermediate during the hydrolysis of ATP. The fraction of enzyme phosphorylated during steady-state ATP hydrolysis was studied as a function of substrate concentration (MgATP), Mg2+ concentration, and pH. The dependence of the fraction of enzyme phosphorylated on the concentration of MgATP is sigmoidal, and the isotherms can be fit with parameters and mechanisms similar to those used to describe ATP hydrolysis. The isotherm is significantly more sigmoidal at pH 5.5 than at pH 6.0, with the limiting percentage (100.mol of phosphate/mol of enzyme) of enzyme phosphorylated being 70% and 6%, respectively, at the two pH values. The maxima in the steady-state rate of ATP hydrolysis occur at higher concentrations of Mg2+ and higher pH than the maxima in the fraction of enzyme phosphorylated. This suggests that the rate-determining step for ATP hydrolysis is different from that for enzyme phosphorylation and the hydrolysis of phosphoenzyme is enhanced by Mg2+ and high pH. The rate of phosphoenzyme formation was investigated with the quenched-flow method, but only a lower bound of 140 s-1 could be obtained for the rate constant at MgATP concentrations greater than 2.5 mM. Since the turnover number for ATP hydrolysis under similar conditions is 14 s-1, the rate-determining step in ATP hydrolysis occurs after enzyme phosphorylation.

Duke Scholars

Published In

J Biol Chem

ISSN

0021-9258

Publication Date

September 25, 1988

Volume

263

Issue

27

Start / End Page

13774 / 13778

Location

United States

Related Subject Headings

  • Vanadates
  • Saccharomyces cerevisiae
  • Proton-Translocating ATPases
  • Phosphoproteins
  • Magnesium
  • Kinetics
  • Hydrolysis
  • Hydrogen-Ion Concentration
  • Cell Membrane
  • Biochemistry & Molecular Biology
 

Citation

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MLA
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Smith, K. E., & Hammes, G. G. (1988). Studies of the phosphoenzyme intermediate of the yeast plasma membrane proton-translocating ATPase. J Biol Chem, 263(27), 13774–13778.
Smith, K. E., and G. G. Hammes. “Studies of the phosphoenzyme intermediate of the yeast plasma membrane proton-translocating ATPase.J Biol Chem 263, no. 27 (September 25, 1988): 13774–78.
Smith, K. E., and G. G. Hammes. “Studies of the phosphoenzyme intermediate of the yeast plasma membrane proton-translocating ATPase.J Biol Chem, vol. 263, no. 27, Sept. 1988, pp. 13774–78.
Smith KE, Hammes GG. Studies of the phosphoenzyme intermediate of the yeast plasma membrane proton-translocating ATPase. J Biol Chem. 1988 Sep 25;263(27):13774–13778.

Published In

J Biol Chem

ISSN

0021-9258

Publication Date

September 25, 1988

Volume

263

Issue

27

Start / End Page

13774 / 13778

Location

United States

Related Subject Headings

  • Vanadates
  • Saccharomyces cerevisiae
  • Proton-Translocating ATPases
  • Phosphoproteins
  • Magnesium
  • Kinetics
  • Hydrolysis
  • Hydrogen-Ion Concentration
  • Cell Membrane
  • Biochemistry & Molecular Biology