Role of lysine 240 in the mechanism of yeast pyruvate kinase catalysis

Site-directed mutagenesis was used to change Lys 240 of yeast pyruvate kina se (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 Mn2+ and Mg2+, respectively. Steady-state fluorescence t itration data suggest that the substrate PEP binds to K240M with the same a ffinity as it does to wild-type YPK. The rate of phosphoryl transfer in K24 0M has been decreased > 1000-fold compared to wild-type YPK. The detritiati on of 3-[H-3]pyruvate catalyzed by YPK occurs at a rate significantly great er than the spontaneous rate. Detritiation of pyruvate by wild-type YPK occ urs as a divalent metal- and FBP-dependent process requiring ATP. There is no detectable detritiation of pyruvate catalyzed by K240M. The solvent deut erium 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 sensit ive step in the PK reaction does not involve Lys 240 and that the enolpyruv ate 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 t rapped with K240M. In K240M, the proton donor exchanges protons with the so lvent at a higher rate relative to turnover than does the proton donor in w ild-type YPK. The pH-rate profile of K240M exhibits the loss of a pK(a) val ue of 8.8 observed with wild-type YPK. The above data and recent crystal st ructure data suggest that Lys 240 interacts with the phosphoryl group of ph osphoenolpyruvate and helps to stabilize the pentavalent phosphate transiti on state during phosphoryl transfer. Phosphoryl transfer is highly coupled to proton transfer, or Lys 240 also affects enolate protonation.