Differential role of four cysteines on the activity of a low M(r) phosphotyrosine protein phosphatase.
In this paper we describe the construction of 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 of 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.
Chiarugi
P
P
Department of Biochemical Sciences, University of Florence, Firenze, Italy.
Marzocchini
R
R
Raugei
G
G
Pazzagli
C
C
Berti
A
A
Camici
G
G
Manao
G
G
Cappugi
G
G
Ramponi
G
G
D018528:ATP-Binding Cassette Transporters; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002417:Cattle; D003545:Cysteine / Q000502:physiology*; D004591:Electrophoresis, Polyacrylamide Gel; D029968:Escherichia coli Proteins; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008320:Maltose / Q000378:metabolism; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D016297:Mutagenesis, Site-Directed; D009596:Nitrophenols / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000378:metabolism; D010957:Plasmids; D017027:Protein Tyrosine Phosphatases / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D009596:Nitrophenols; D009943:Organophosphorus Compounds; D011993:Recombinant Fusion Proteins; C461599:maltose transport system, E coli; C008644:nitrophenylphosphate; D008320:Maltose; D017027:Protein Tyrosine Phosphatases; D003545:Cysteine