Characterization of active-site mutants of Schizosaccharomyces pombe phosphoglycerate mutase - Elucidation of the roles of amino acids involved in substrate binding and catalysis

The roles of a number of amino acids present at the active site of the mono meric phosphoglycerate mutase from the fission yeast Schizosaccharomyces po mbe have been explored by site-directed mutagenesis. The amino acids examin ed could be divided broadly into those presumed from previous related struc tural studies to be important in the catalytic process (R14, S62 and E93) a nd those thought to be important in substrate binding (R94, R120 and R121). Most of these residues have not previously been studied by site-directed m utagenesis. All the mutants except R14 were expressed in an engineered null strain of Saccharomyces cerevisiae (S150-gpm::HIS) in good yield. The R14Q mutant was expressed in good yield in the transformed AH22 strain of S. ce revisiae. The S62A mutant was markedly unstable, preventing purification. T he various mutants were purified to homogeneity and characterized in terms of kinetic parameters, CD and fluorescence spectra, stability towards denat uration by guanidinium chloride, and stability of phosphorylated enzyme int ermediate. In addition, the binding of substrate (3-phosphoglycerate) to wi ld-type, E93D and R120,121Q enzymes was measured by isothermal titration ca lorimetry. The results provide evidence for the proposed roles of each of t hese amino acids in the catalytic cycle and in substrate binding, and will support the current investigation of the structure and dynamics of the enzy me using multidimensional NMR techniques.