Proton transfer and the catalytic mechanism of yeast pyruvate kinase

The identification of the proton donor to enolpyruvate by yeast pyruvate kinase (YPK) was addressed.; The T298S, T298C, and T298A mutants of YPK were constructed, purified and investigated by kinetics, ligand binding, solvent isotope effects, and proton inventories. The results indicate that T298 is not the ultimate proton donor to enolpyruvate but water in a channel into the active site serves this function. T298 plays a role in a late step in the catalytic mechanism that involves proton transfer. One water in this channel is coordinated to the enzyme-bound divalent metal making this reaction cation dependent. T298 appears to interact with the terminal water molecule in the proton circuit. The residue at position 298 affects the pKa of water in the channel and therefore its reactivity. These results suggest that Thr298 is important in the interaction of the substrate PEP with the enzyme and in the proper orientation of PEP for catalysis.; Temperature dependence of the water proton relaxation rates of the PK-Mn 2+-PEP and PK-Mn2+-PEP-FBP complexes of wild type YPK and T298S and T298A mutants indicates that the enzyme-bound Mn2+ is isolated and distant from bulk water. Although the details of these interactions are not clear, it is evident that the relaxation effects are long-distance effects. The results are consistent with a water network around the enzyme-bound Mn2+ at the active site that extends to the bulk solvent. The proposed catalytically important water network in the active site of pyruvate kinase is supported by the X-ray structure of the bis(Mg2+)-ATP-oxalate complex of rabbit muscle PK at 2.1 A resolution, where specific water molecules are indicated (Larsen et al., 1998).; The interaction of the monovalent cation with wild type YPK and with the T298S, T298C, and T298A mutants was investigated by 205Tl + NMR to monitor possible structural alterations at the active site by mutation of T298. The Tl+-Mn2+ distances at the active site of YPK were calculated from the paramagnetic contribution of Mn2+ to the longitudinal relaxation rates of YPK-bound 205Tl+. The results suggest conformational alterations of the substrate and of the cations at the site of phosphoryl transfer upon mutation of T298. The substrate conformational changes may, in part, explain the alteration in kcat and k cat/Km,PEP observed with the T298 mutants.