title: Development of a mutagenesis, expression and purification system for yeast phosphoglycerate mutase. Investigation of the role of active-site His181.
abstract: A system has been developed to allow the convenient production, expression and purification of site-directed mutants of the enzyme phosphoglycerate mutase from Saccharomyces cerevisiae. This enzyme is well characterised; both the amino acid sequence and crystal structure have been determined and a reaction mechanism has been proposed. However, the molecular basis for catalysis remains poorly understood, with only circumstantial evidence for the roles of most of the active site residues other than His8, which is phosphorylated during the reaction cycle. A vector/host expression system has been designed which allows recombinant forms of phosphoglycerate mutase to be efficiently expressed in yeast with no background wild-type activity. A simple one-column purification protocol typically yields 30 mg pure enzyme/1 l of culture. The active-site residue, His181, which is thought to be involved in proton transfer during the catalytic cycle, has been mutated to an alanine. The resultant mutant has been purified and characterised. Kinetic analysis shows a large decrease (1.6 x 10(4)) in the catalytic efficiency, and an 11-fold increase in the Km for the cofactor 2,3-bisphosphoglycerate. These observations are consistent with an integral role for His181 in the reaction mechanism of phosphoglycerate mutase, probably as a general acid or base.
authors.affiliation: Department of Biochemistry, University of Edinburgh, Scotland.
authors.forename: M F
authors.initials: MF
authors.lastname: White
authors.forename: L A
authors.initials: LA
authors.lastname: Fothergill-Gilmore
mesh_terms: D001665:Binding Sites; D001731:Bisphosphoglycerate Mutase / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular; D006639:Histidine / Q000737:chemistry; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011994:Recombinant Proteins; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
chemical_list: D011994:Recombinant Proteins; D006639:Histidine; D001731:Bisphosphoglycerate Mutase