Phosphoglycerate mutase from Schizosaccharomyces pombe: development of an expression system and characterisation of three histidine mutants of the enzyme.
The small, monomeric, phosphoglycerate mutase (PGAM) from Schizosaccharomyces pombe has been overexpressed in a strain of Saccharomyces cerevisiae 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 in which histidine residues in S. pombe PGAM have been substituted by glutamine have been purified and characterised. Two mutants (H151Q and H196Q) have kinetic and structural properties very similar to wild-type enzyme, consistent with the proposed location of these (non-conserved) histidines on the surface of the enzyme. The third mutant (H163Q) involving a histidine thought to be part of the active site has greatly reduced mutase and phosphatase activities. Mass spectrometry shows that the phosphorylated form of the H163Q is several 100-times more stable 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.
Nairn
J
J
Department of Biological and Molecular Sciences, University of Stirling, UK.
Price
N C
NC
Kelly
S M
SM
Rigden
D
D
Fothergill-Gilmore
L A
LA
Krell
T
T
D001483:Base Sequence; D002942:Circular Dichroism; D006639:Histidine / Q000235:genetics; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010736:Phosphoglycerate Mutase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012568:Schizosaccharomyces / Q000201:enzymology* / Q000235:genetics; D013050:Spectrometry, Fluorescence
D011994:Recombinant Proteins; D006639:Histidine; D010736:Phosphoglycerate Mutase