Characterisation of active-site mutants of Schizosaccharomyces pombe phosphoglycerate mutase

Citation

Nairn J, Duncan D, Price NE, Kelly SM, Fothergill-Gilmore LA, Uhrinova S, Barlow PN, Rigden DJ & Price NC (2000) Characterisation of active-site mutants of Schizosaccharomyces pombe phosphoglycerate mutase. European Journal of Biochemistry, 267 (24), pp. 7065-7074. https://doi.org/10.1046/j.1432-1327.2000.01802.x

Abstract
The roles of a number of amino acids present at the active site of the monomeric phosphoglycerate mutase from the fission yeast Schizosaccharomyces pombe have been explored by site-directed mutagenesis. The amino acids examined could be divided broadly into those presumed from previous related structural studies to be important in the catalytic process (R14, S62 and E93) and those thought to be important in substrate binding (R94, R120 and R121). Most of these residues have not previously been studied by site-directed mutagenesis. 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. cerevisiae. The S62A mutant was markedly unstable, preventing purification. The various mutants were purified to homogeneity and characterized in terms of kinetic parameters, CD and fluorescence spectra, stability towards denaturation by guanidinium chloride, and stability of phosphorylated enzyme intermediate. In addition, the binding of substrate (3-phosphoglycerate) to wild-type, E93D and R120,121Q enzymes was measured by isothermal titration calorimetry. The results provide evidence for the proposed roles of each of these amino acids in the catalytic cycle and in substrate binding, and will support the current investigation of the structure and dynamics of the enzyme using multidimensional NMR techniques

Keywords
catalytic mechanism; phosphoglycerate mutase; site-directed mutagenesis; protein stability; substrate binding; Yeast; Amino acids Metabolism; Mutagenesis

Journal
European Journal of Biochemistry: Volume 267, Issue 24