title: Lack of evidence for a role of Cys-138 as a base catalyst in the skeletal muscle 6-phosphofructo-2-kinase reaction.
abstract: The role of Cys-138 in the catalysis of the skeletal muscle 6-phosphofructo-2-kinase reaction was investigated by mutating this residue to serine, glutamine and alanine, expressing the mutants in E. coli with a T7 RNA polymerase-based expression system, and analyzing their kinetic properties. The Cys138Ala mutant had greatly diminished activity, while the Cys138Ser and Cys138Gln mutants had maximal velocities 2-3 fold higher than the wild-type enzyme. It was concluded that Cys-138 does not act as a base catalyst in the kinase reaction, but that it plays a significant structural role in the enzyme's active site.
authors.affiliation: Department of Physiology and Biophysics, State University of New York, Stony Brook 11794-8661.
authors.forename: I J
authors.initials: IJ
authors.lastname: Kurland
authors.forename: M R
authors.initials: MR
authors.lastname: el-Maghrabi
authors.forename: S J
authors.initials: SJ
authors.lastname: Pilkis
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D003545:Cysteine; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D009132:Muscles / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010732:Phosphofructokinase-1 / Q000235:genetics / Q000378:metabolism*; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D010732:Phosphofructokinase-1; D003545:Cysteine