Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 278, Issue 15, Pages 12660-12671Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M300257200
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- NIDDK NIH HHS [DK 17049] Funding Source: Medline
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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,K-PEP. Mn2+-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-Mn2+ is cooperative. The pH dependence of k(cat) for T298A indicates the loss of pK(alpha,2) = 6.4-6.9. Thr-298 affects the ionization (pK(alpha) approximate to6.5) 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,K-PEP. Fractionation factors are metal-dependent and significantly <1. The data suggest that a water molecule in a water channel is the direct proton donor to enolpyruvate and that Thr-298 affects a late step in catalysis.
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