title: Aspartic-129 is an essential residue in the catalytic mechanism of the low M(r) phosphotyrosine protein phosphatase.
abstract: The crystal structure of the bovine liver low M(r) phosphotyrosine protein phosphatase suggests the involvement of aspartic acid-129 in enzyme catalysis. The Asp-129 to alanine mutant has been prepared by oligonucleotide-directed mutagenesis of a synthetic gene coding for the enzyme. The purified mutant elicited an highly reduced specific activity (about 0.04% of the activity of the wild-type) and a native-like fold, as judged by 1H NMR spectroscopy. The kinetic analysis revealed that the mutant is able to bind the substrate and a competitive inhibitor, such as inorganic phosphate. Moreover, trapping experiments demonstrated it maintains the ability to form the E-P covalent complex. The Asp-129 to alanine mutant shows extremely reduced enzyme phosphorylation (k2) and dephosphorylation (k3) kinetic constant values as compared to the wild-type enzyme. The data reported indicate that aspartic acid-129 is likely to be involved both in the first step and in the rate-limiting step of the catalytic mechanism, i.e. the nucleophilic attack of the phosphorylated intermediate.
authors.affiliation: Department of Biochemical Sciences, University of Florence, Italy.
authors.forename: N
authors.initials: N
authors.lastname: Taddei
authors.forename: P
authors.initials: P
authors.lastname: Chiarugi
authors.forename: P
authors.initials: P
authors.lastname: Cirri
authors.forename: T
authors.initials: T
authors.lastname: Fiaschi
authors.forename: M
authors.initials: M
authors.lastname: Stefani
authors.forename: G
authors.initials: G
authors.lastname: Camici
authors.forename: G
authors.initials: G
authors.lastname: Raugei
authors.forename: G
authors.initials: G
authors.lastname: Ramponi
mesh_terms: D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry; D001483:Base Sequence; D002417:Cattle; D017931:DNA Primers / Q000737:chemistry; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism*; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship
chemical_list: D017931:DNA Primers; D011994:Recombinant Proteins; D001224:Aspartic Acid; D017027:Protein Tyrosine Phosphatases