THE ROLE OF CYS12, CYS17 AND ARG18 IN THE CATALYTIC MECHANISM OF LOW-MR CYTOSOLIC PHOSPHOTYROSINE PROTEIN PHOSPHATASE

Low-M(r) phosphotyrosine protein phosphatase (PTPase), previously know n as low-M(r) acid phosphatase, catalyzes the in-vitro hydrolysis of t yrosine 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-p hosphate hydrolysis catalyzed by the enzyme was compared to the known mechanism of p-nitrophenyl-phosphate hydrolysis. The results demonstra ted that both hydrolytic processes proceed through common enzyme-catal yzed mechanisms. Nevertheless, the performed phosphoenzyme-trapping ex periments enable us to identify Cys12 as the active-site residue that performs the nucleophilic attack at the phosphorus atom of the substra te to produce a phosphoenzyme covalent intermediate. In addition, whil e the role of Cys17 in the substrate binding was confirmed, its partic ipation a second time in the step that involves the Cys12 dephosphoryl ation was suggested by the results of phosphoenzyme-trapping experimen ts. The participation of Arg18 in the substrate-binding site was demon strated by site-directed mutagenesis that produced the conservative Ly s18 and the non-conservative Met18 mutants. Both these mutants were al most inactive and not able to bind the substrate and a competitive inh ibitor. Furthermore, phosphoenzyme-trapping experiments clearly exclud ed 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 ph osphoenzyme covalent intermediate.