Differential role of four cysteines on the activity of a low M r phosphotyrosine protein phosphatase

FEBS Letters

Raugei, Giovanni¹  Ramponi, Giampietro¹  Camici, Guido¹  Cappugi, Gianni¹  Marzocchini, Riccardo¹  Pazzagli, Claudia¹  Berti, Andrea¹  Manao, Gianpaolo¹  Chiarugi, Paola¹ 

[1] Department of Biochemical Sciences, University of Florence, Viale Morgagni 50, 50134 Firenze, Italy

关键词: Acid phosphatase;    Phosphotyrosyl protein phosphatase;    Fusion protein;    Site-directed mutagenesis;    Active site;   

DOI  :  10.1016/0014-5793(92)81134-8

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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.

【 授权许可】

Unknown