PHOSPHOGLYCERATE MUTASE FROM SCHIZOSACCHAROMYCES-POMBE - DEVELOPMENT OF AN EXPRESSION SYSTEM AND CHARACTERIZATION OF 3 HISTIDINE MUTANTS OFTHE ENZYME

The small, monomeric, phosphoglycerate mutase (PGAM) from Schizosaccha romyces pombe has been overexpressed in a strain of Saccharomyces cere visiae in which the gene encoding PGAM has been deleted, with a yield of purified enzyme of 10-15 mg per litre cell culture. Three mutants i n which histidine residues in S. pombe PGAM have been substituted by g lutamine have been purified and characterised. Two mutants (H151Q and H196Q) have kinetic and structural properties very similar to wild-typ e enzyme, consistent with the proposed location of these (non-conserve d) histidines on the surface of the enzyme. The third mutant (H163Q) i nvolving a histidine thought to be part of the active site has greatly reduced mutase and phosphatase activities. Mass spectrometry shows th at the phosphorylated form of the H163Q is several 100-times more stab le towards hydrolysis than the phosphorylated form of wild-type enzyme . The H163Q mutant appears to be structurally quite distinct from wild -type enzyme. 600 MHz 1D proton NMR spectra of good quality have been obtained for wild-type enzyme and the H151Q and H196Q mutants.