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Differential role of four cysteines on the activity of a low M r phosphotyrosine protein phosphatase
Author(s) -
Chiarugi Paola,
Marzocchini Riccardo,
Raugei Giovanni,
Pazzagli Claudia,
Berti Andrea,
Camici Guido,
Manao Gianpaolo,
Cappugi Gianni,
Ramponi Giampietro
Publication year - 1992
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(92)81134-8
Subject(s) - maltose binding protein , mutant , phosphatase , biochemistry , site directed mutagenesis , fusion protein , enzyme , mutant protein , mutagenesis , chemistry , protein tyrosine phosphatase , biology , microbiology and biotechnology , recombinant dna , gene
In this paper we describe the construction or five mutants of a bovine liver low M r phosphotyrosine protein phosphatase (PTPase) expressed as a fusion protein with the maltose binding protein in E. coli . Almost no changes in the kinetic parameters were observed in the fusion protein with respect to the native PTPase. Using oligonucleotide‐directed mutagenesis Cys‐17, Cys‐62 and Cys‐145 were converted to Ser while Cys‐12 was converted to both Ser and Ala. The kinetic properties of the mutants, using p ‐nitrophenyl phosphate as substrate, were compared with those of the normal protein fused with the maltose binding protein or E. coli ; both of the Cys‐12 mutants showed a complete loss of enzymatic activity while the specific activity of the Cys‐17 mutant was greatly decreased (200‐fold). The Cys‐62 mutant showed a 2.5‐fold decrease in specific activity, while the Cys‐145 mutant remained almost unchanged. These data confirm the involvement of Cys‐12 and Cys‐17 in the catalytic site and suggest that Cys‐62 and Cys‐145 mutations may destabilise the structure of the enzyme.