Spectral studies of the interactions of Escherichia coli alkaline phosphatase with 4-(4-aminophenylazo)-phenylarsonic acid.
Escherichia coli alkaline phosphatase (EC 3.1.3.1) is reversibly inhibited by a variety of phenylarsonic acids, including some N-haloacetylated derivatives. The inhibition is of the competitive type, and Ki values are reported. The action on the enzyme of one of the arsonate inhibitors, the azo dye, 4-(4-aminophenylazo)-phenylarsonic acid was studied in detail, using spectrophotometric and kinetic methods. The azo dye binds more strongly to E. coli alkaline phosphatase than do the other arsonates. Spectrophotometric titration indicates the presence of a single, strong dye-binding site on the enzyme dimer molecule in the concentration range covered. In 0.1 M Tris - HCl buffer pH 8.0, 25 degrees C K diss for the dye - enzyme complex is 1.50 - 10(-5) M as determined by spectrophotometric titration. This value is in good agreement with the Ki = 1.30 - 10(-5) M obtained from kinetic measurements. The dye can be displaced from alkaline phosphatase by phosphate and competitive inhibitor 2-aminoethyl phosphonate. These results indicate that the dye binds with its arsonic acid group to the anion binding site of the active site of the enzyme. The binding of the dye to the native enzyme is associated with a red shift in the visible spectrum of the dye. It seems that the aromatic portion of the dye interacts with a hydrophobic region close to the anion binding site. The spectrum of the dye is not changed in the presence of the apoenzyme. When zinc is added to an apoenzyme-dye solution, the spectral changes of the dye depend on both the ratio of zinc per apoenzyme and the pH. The presence of Mg2+ had no effect on the observed phenomenon.
D000469:Alkaline Phosphatase / Q000037:antagonists & inhibitors; D001152:Arsenicals; D001391:Azo Compounds; D001665:Binding Sites; D001667:Binding, Competitive; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D010710:Phosphates; D011485:Protein Binding; D011487:Protein Conformation; D013053:Spectrophotometry; D013329:Structure-Activity Relationship; D015032:Zinc
D001152:Arsenicals; D001391:Azo Compounds; D010710:Phosphates; D000469:Alkaline Phosphatase; D015032:Zinc