title: Yeast phosphoglycerate kinase: investigation of catalytic function by site-directed mutagenesis.
abstract: A salt link buried in the domain interface of phosphoglycerate kinase has been implicated as being important in controlling the conformational transition from the open, or substrate-binding, to the closed, or catalytically competent, form of the enzyme. The residues contributing to the salt link are remote from the active site, but are connected to the substrate-binding sites through strands of beta-sheet. It has been suggested that these residues may also mediate sulphate and anion activation. These assumptions have been tested by examining the properties of a site-directed mutant (histidine-388----glutamine-388). The expression and overall structural integrity of the mutant, produced in yeast from a multicopy plasmid, remains essentially unaltered from the wild-type enzyme. However, the mutant enzyme has a kcat. reduced by 5-fold. The Km for ATP is lowered by 3-fold, and the Km for 3-phosphoglycerate is unaffected. The effects of sulphate on activity over a wide range of substrate concentrations appear to be the same for both the mutant and wild-type enzymes. These results lead to a reappraisal of the mechanistic role of the inter-domain histidine-glutamate interaction, as well as a refinement of the kinetic model of the enzyme.
authors.forename: C A
authors.initials: CA
authors.lastname: Wilson
authors.forename: N
authors.initials: N
authors.lastname: Hardman
authors.forename: L A
authors.initials: LA
authors.lastname: Fothergill-Gilmore
authors.forename: S J
authors.initials: SJ
authors.lastname: Gamblin
authors.forename: H C
authors.initials: HC
authors.lastname: Watson
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D005786:Gene Expression Regulation; D009154:Mutation; D009841:Oligonucleotides / Q000494:pharmacology; D010735:Phosphoglycerate Kinase / Q000235:genetics / Q000378:metabolism*; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics
chemical_list: D009841:Oligonucleotides; D010735:Phosphoglycerate Kinase