Aspartic-129 is an essential residue in the catalytic mechanism of the low M(r) phosphotyrosine protein phosphatase.
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.
Taddei
N
N
Department of Biochemical Sciences, University of Florence, Italy.
Chiarugi
P
P
Cirri
P
P
Fiaschi
T
T
Stefani
M
M
Camici
G
G
Raugei
G
G
Ramponi
G
G
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
D017931:DNA Primers; D011994:Recombinant Proteins; D001224:Aspartic Acid; D017027:Protein Tyrosine Phosphatases