The role of Cys12, Cys17 and Arg18 in the catalytic mechanism of low-M(r) cytosolic phosphotyrosine protein phosphatase.
Low-M(r) phosphotyrosine protein phosphatase (PTPase), previously known as low-M(r) acid phosphatase, catalyzes the in-vitro hydrolysis of tyrosine phosphorylated proteins, low-M(r) aryl phosphates and natural and synthetic acyl phosphates. Its activity on Ser/Thr-phosphorylated proteins and on most alkyl phosphates is very poor. In this study the mechanism of benzoyl-phosphate hydrolysis was studied by means of non-mutated and mutated PTPase fusion proteins. The mechanism of benzoyl-phosphate hydrolysis catalyzed by the enzyme was compared to the known mechanism of p-nitrophenyl-phosphate hydrolysis. The results demonstrated that both hydrolytic processes proceed through common enzyme-catalyzed mechanisms. Nevertheless, the performed phosphoenzyme-trapping experiments enable us to identify Cys12 as the active-site residue that performs the nucleophilic attack at the phosphorus atom of the substrate to produce a phosphoenzyme covalent intermediate. In addition, while the role of Cys17 in the substrate binding was confirmed, its participation a second time in the step that involves the Cys12 dephosphorylation was suggested by the results of phosphoenzyme-trapping experiments. The participation of Arg18 in the substrate-binding site was demonstrated by site-directed mutagenesis that produced the conservative Lys18 and the non-conservative Met18 mutants. Both these mutants were almost inactive and not able to bind the substrate and a competitive inhibitor. Furthermore, phosphoenzyme-trapping experiments clearly excluded that Cys62 and Cys145 (that were indicated by another laboratory to be involved in the active site of the enzyme as powerful nucleophilic agents) are the residues directly involved in the formation of the phosphoenzyme covalent intermediate.
Cirri
P
P
Department of Biochemical Sciences, University of Florence, Italy.
Chiarugi
P
P
Camici
G
G
Manao
G
G
Raugei
G
G
Cappugi
G
G
Ramponi
G
G
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000235:genetics / Q000378:metabolism; D001565:Benzoates / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D002417:Cattle; D003545:Cysteine / Q000031:analogs & derivatives / Q000235:genetics / Q000378:metabolism; D003600:Cytosol / Q000201:enzymology; D004252:DNA Mutational Analysis; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008954:Models, Biological; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D017027:Protein Tyrosine Phosphatases / Q000235:genetics* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D001565:Benzoates; D011993:Recombinant Fusion Proteins; C012217:benzoyl phosphate; D001120:Arginine; D017027:Protein Tyrosine Phosphatases; D003545:Cysteine