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Site‐directed mutagenesis of histidine 62 in the ‘basic patch’ region of yeast phosphoglycerate kinase
Author(s) -
Fairbrother Wayne J.,
Hall Len,
Littlechild Jennifer A.,
Walker Philip A.,
Watson Herman C.,
Williams Robert J.P.
Publication year - 1989
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(89)81665-5
Subject(s) - phosphoglycerate kinase , site directed mutagenesis , mutant , mutagenesis , biochemistry , histidine , yeast , enzyme , wild type , chemistry , glutamine , residue (chemistry) , binding site , biology , stereochemistry , microbiology and biotechnology , amino acid , gene
Site‐directed mutagenesis has been used to produce a mutant form of yeast phosphoglycerate kinase (PGK) in which the ‘basic patch’ residue His 62 has been replaced by a glutamine residue. Using 1 H‐NMR spectroscopy, it was found that 3‐phosphoglycerate (3‐PG) binding to the mutant protein induces the same conformational effects as for wild‐type PGK, although the affinity was reduced by 2‐ to 3‐fold. Kinetic studies show both K m for 3‐PG and V max to be increased by ~ 2‐fold relative to the wild‐type enzyme. These data are consistent with the suggestion that His 62 assists in the binding of the substrate to the enzyme.