The role of His66 and His72 in the reaction mechanism of bovine liver low-M(r) phosphotyrosine protein phosphatase.
Site-directed mutagenesis of a synthetic gene coding for low-M(r) phosphotyrosine protein phosphatase from bovine liver has been carried out. The two histidine residues in the enzyme have been mutated to glutamine; both single and double mutants were produced. The mutated and non-mutated sequences have been expressed in Escherichia coli as fusion proteins, in which the low-M(r) phosphotyrosine protein phosphatase was linked to the C-terminal end of the maltose-binding protein. The fusion enzymes were easily purified by single-step affinity chromatography. The mutants were studied for their kinetic properties. Both single mutants showed decreased kcat. values (30 and 7% residual activities for His66 and His72 respectively), and alterations of the Ki values relative to four-competitive inhibitors were observed. The kinetic mechanism of p-nitrophenyl phosphate hydrolysis in the presence of both single mutants was determined and compared with that of the non-mutated enzyme. The rate-determining step of the catalytic process of the His66-->Gln mutant was the same as that found for non-mutated enzyme, whereas for the His72-->Gln mutant, both the kinetic constant of the step that causes the formation of a phosphoenzyme covalent intermediate, and the kinetic constant of the step that causes the dephosphorylation of the enzyme covalent intermediate, determined the kcat. value. This observation was confirmed by phosphoenzyme covalent intermediate trapping experiments. The participation of both histidine residues (His66 and His72) at the active site is strongly suggested by the results of diethyl pyrocarbonate inactivation of both single mutants, each containing a single histidine residue. Both mutants are completely inactivated by diethyl pyrocarbonate treatment; the competitive inhibitor Pi protects both mutants from inactivation. The His66/His72 double mutant was completely inactive.
Chiarugi
P
P
Dipartimento di Scienze Biochimiche, Università di Firenze, Italy.
Cirri
P
P
Camici
G
G
Manao
G
G
Fiaschi
T
T
Raugei
G
G
Cappugi
G
G
Ramponi
G
G
D000595:Amino Acid Sequence; D000818:Animals; D005075:Biological Evolution; D002384:Catalysis; D002417:Cattle; D017124:Conserved Sequence; D004047:Diethyl Pyrocarbonate; D006639:Histidine / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D017027:Protein Tyrosine Phosphatases / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D011993:Recombinant Fusion Proteins; D006639:Histidine; D017027:Protein Tyrosine Phosphatases; D004047:Diethyl Pyrocarbonate