title: Kinetic and allosteric consequences of mutations in the subunit and domain interfaces and the allosteric site of yeast pyruvate kinase.
abstract: The mechanism by which pyruvate kinase (PK) is allosterically activated by fructose-1,6-bisphosphate (FBP) is poorly understood. To identify residues key to allostery of yeast PK, a point mutation strategy was used. T403E and R459Q mutations in the FBP binding site caused reduced FBP affinity. Introducing positive charges at the 403, 458, and 406 positions in the FBP binding site had little consequence. The mutation Q299N in the A [bond] A subunit interface caused the enzyme response to ADP to be sensitive to FBP. The T311M A [bond] A interface mutant has a decreased affinity for PEP and FBP, and is dependent on FBP for activity. The R369A mutation in the C [bond] C interface only moderately influenced allostery. Creating an E392A mutation in the C [bond] C subunit interface eliminated all cooperativity and allosteric regulation. None of the seven A [bond] C domain interface mutations altered allostery. A model that includes a central role for E392 in allosteric regulation of yeast PK is proposed.
authors.affiliation: Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, Oklahoma 74078, USA. aron@hugo.tamu.edu
authors.forename: Aron W
authors.initials: AW
authors.lastname: Fenton
authors.forename: James B
authors.initials: JB
authors.lastname: Blair
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism; D000495:Allosteric Site; D001483:Base Sequence; D001665:Binding Sites; D018360:Crystallography, X-Ray; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005635:Fructosediphosphates / Q000737:chemistry / Q000235:genetics; D007700:Kinetics; D008954:Models, Biological; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D011770:Pyruvate Kinase / Q000737:chemistry* / Q000235:genetics*; D013050:Spectrometry, Fluorescence
chemical_list: D005635:Fructosediphosphates; D000244:Adenosine Diphosphate; D011770:Pyruvate Kinase; C029063:fructose-1,6-diphosphate