Role of lysine 240 in the mechanism of yeast pyruvate kinase catalysis.
Site-directed mutagenesis was used to change Lys 240 of yeast pyruvate kinase (Lys 269 in muscle PK) to Met. K240M has an absolute requirement for FBP for catalysis. K240M is 100- and 1000-fold less active than wild-type YPK in the presence of Mn(2+) and Mg(2+), respectively. Steady-state fluorescence titration data suggest that the substrate PEP binds to K240M with the same affinity as it does to wild-type YPK. The rate of phosphoryl transfer in K240M has been decreased >1000-fold compared to wild-type YPK. The detritiation of 3-[(3)H]pyruvate catalyzed by YPK occurs at a rate significantly greater than the spontaneous rate. Detritiation of pyruvate by wild-type YPK occurs as a divalent metal- and FBP-dependent process requiring ATP. There is no detectable detritiation of pyruvate catalyzed by K240M. The solvent deuterium isotope effect on k(cat) is 2.7 +/- 0.2 and 1.6 +/- 0.1 for the wild type and for K240M YPK, respectively. This suggests that the isotope sensitive step in the PK reaction does not involve Lys 240 and that the enolpyruvate intermediate is still protonated by K240M. Isotope trapping was used to characterize enolpyruvate protonation by K240M. While there was enrichment of the methyl protons of pyruvate from labeled solvent formed by catalysis with muscle PK and wild-type YPK, only background levels of tritium were trapped with K240M. In K240M, the proton donor exchanges protons with the solvent at a higher rate relative to turnover than does the proton donor in wild-type YPK. The pH-rate profile of K240M exhibits the loss of a pK(a) value of 8. 8 observed with wild-type YPK. The above data and recent crystal structure data suggest that Lys 240 interacts with the phosphoryl group of phosphoenolpyruvate and helps to stabilize the pentavalent phosphate transition state during phosphoryl transfer. Phosphoryl transfer is highly coupled to proton transfer, or Lys 240 also affects enolate protonation.
Department of Chemistry and Biochemistry, University of Notre Dame, Indiana 46556, USA.
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D002384:Catalysis; D002455:Cell Division / Q000235:genetics; D017666:Deuterium Oxide; D004579:Electron Transport; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008239:Lysine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008715:Methionine / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D011522:Protons; D011770:Pyruvate Kinase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D019289:Pyruvic Acid / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D012997:Solvents; D014316:Tritium
D011522:Protons; D011994:Recombinant Proteins; D012997:Solvents; D014316:Tritium; D019289:Pyruvic Acid; D008715:Methionine; D011770:Pyruvate Kinase; D017666:Deuterium Oxide; D008239:Lysine