The proton transfer step catalyzed by yeast pyruvate kinase

The Journal of Biological Chemistry
Delia Susan-Resiga, T Nowak


The nature of the proton donor to the C-3 of the enolate of pyruvate, the intermediate in the reaction catalyzed by yeast pyruvate kinase, was investigated by site-directed mutagenesis and physical and kinetic analyses. Thr-298 is correctly located to function as the proton donor. T298S and T298A were constructed and purified. Both mutants are catalytically active with a decrease in k(cat) and k(cat)/K(m)(,PEP). Mn(2+)-activated T298S and T298A do not exhibit homotropic kinetic cooperativity with phosphoenolpyruvate (PEP) in the absence of fructose 1,6-bisphosphate, although PEP binding to enzyme-Mn(2+) is cooperative. The pH dependence of k(cat) for T298A indicates the loss of pK(a)(,2) = 6.4-6.9. Thr-298 affects the ionization (pK(a) approximately 6.5) responsible for modulation of k(cat). Fluorescence studies show altered dissociation constants of ligands to each enzyme complex upon Thr-298 mutations. The rates of the phosphoryl transfer and proton transfer steps in the pyruvate kinase-catalyzed reaction are altered; pyruvate enolization is affected to a greater extent. Proton inventory studies demonstrate solvent isotope effects on k(cat) and k(cat)/K(m)(,PEP). Fractionation factors are metal-dependent and significantly <1....Continue Reading


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Jun 10, 2003·The Journal of Biological Chemistry·Sandrine GulbertiMohamed Ouzzine
Oct 15, 2019·Journal of Biomolecular Structure & Dynamics·J Dennis PollackDennis K Pearl


Jun 7, 1976·Biochimica Et Biophysica Acta·D J Leblond, J L Robinson
Jun 7, 1976·Biochimica Et Biophysica Acta·S R Ford, J L Robinson
Jan 22, 1991·Biochemistry·I A RoseJ V Warms
Jan 22, 1991·Biochemistry·S H SeeholzerI A Rose
Dec 12, 1989·Biochemistry·I A Rose, D J Kuo
Jan 1, 1983·Journal of Bacteriology·H ItoA Kimura
Mar 18, 1997·Proceedings of the National Academy of Sciences of the United States of America·T I OpreaA E Garcia
Jul 22, 1999·Biochemistry·T J BollenbachT Nowak
Jun 29, 2000·Annual Review of Biochemistry·D W Christianson, J D Cox
Dec 1, 1958·Archives of Biochemistry and Biophysics·A TIETZ, S OCHOA

Related Concepts

Cations, Divalent
Proteins, Recombinant DNA
Mutagenesis, Site-Directed
Pyruvate Measurement
Pyruvate Kinase
Fluorescence Spectroscopy
Water channel
Pyruvate Kinase Measurement

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