nitrophenols and beta-naphthyl-phosphate

Uric acid inhibited 50% of the activity of bovine kidney low molecular mass phosphotyrosine protein phosphatase at concentrations of 1.0, 0.4, 1.3, and 0.2 mM, respectively for p-nitrophenyl phosphate (p-NPP), flavine mononucleotide, beta-naphthyl phosphate and tyrosine phosphate (Tyr-P) as substrates. The mixed type inhibition of p-NPP hydrolysis was fully reversible, with Kic and Kiu values of 0.4 and 1.1 mM, respectively; the inhibition by uric acid shifted the pH optimum from 5.0 to 6.5. When Tyr-P was the substrate, competitive inhibition was observed with a Ki value of 0.05 mM. Inhibition studies by uric acid in the presence of thiol compounds, and preincubation studies in the presence of inorganic phosphate suggest that the interaction of uric acid with the enzyme occurred at the active site, but did not involve SH residues, and that the mechanism of inhibition depended on the structure of the substrates.

Topics: Animals; Binding Sites; Binding, Competitive; Cattle; Enzyme Inhibitors; Flavin Mononucleotide; Kidney; Kinetics; Naphthalenes; Nitrophenols; Organophosphates; Organophosphorus Compounds; Phosphates; Protein Tyrosine Phosphatases; Substrate Specificity; Sulfhydryl Compounds; Tyrosine; Uric Acid

A low molecular weight bovine kidney acid phosphatase, electrophoretically homogeneous and with a relative molecular mass of 17.8 kDa, was used in this work. Among the various substrates tested, FMN was found to be the most effective, at pH 7.0. Distinct activation energy values were obtained for p-nitrophenyl phosphate- (45.44 kJ mol-1) and flavin mononucleotide- (28.60 kJ mol-1) hydrolysis reactions. The FMN hydrolysis was strongly inhibited by Cu2 and pCMB, but activated by guanosine. Pyridoxal-phosphate and vanadate were competitive inhibitors for the FMN-dependent reaction.

Topics: Acid Phosphatase; Animals; Cattle; Chloromercuribenzoates; Copper; Electrophoresis, Polyacrylamide Gel; Flavin Mononucleotide; Guanosine; Hydrogen-Ion Concentration; Hydrolysis; Indicators and Reagents; Kidney; Kinetics; Molecular Weight; Naphthalenes; Nitrophenols; Organophosphates; Organophosphorus Compounds; p-Chloromercuribenzoic Acid; Phosphotyrosine; Pyridoxal Phosphate; Substrate Specificity; Vanadates