Irreversible inactivation of purple acid phosphatase by hydrogen peroxide and ascorbate

摘要

Treatment of the Cu(II)-Fe(III) derivative of pig allantoic fluid acid phosphatase with hydrogen peroxide caused irreversible inactivation of the enzyme and loss of half of the intensity of the visible absorption spectrum. Phosphate, a competitive inhibitor, protected against this inactivation, suggesting that it occurred as a result of a reaction at the active site. The native Fe(II)-Fe(III) enzyme was irreversibly inactivated by H_2O_2 to a much smaller extent that the Cu(II)-Fe(III) derivative, whereas the Zn(II)-Fe(III) derivative was stable to H_2O_2 treatment. The rates of inactivation of the Cu(II)-Fe(III) and Fe(II)-Fe(III) enzymes in the presence of H_2O_2 were increased by addition of ascorbate. These results suggest involvement of a Fenton-type reaction, generating hydroxyl radicals which react with essential active site groups. Experiments carried out on the Fe(II)-Fe(III) enzyme showed that irreversible inactivation by H_2O_2 in the presence of ascorbate obeyed pseudo first-order kinetics. A plot of k_(obs) for this reaction against H_2O_2 concentration (at saturating ascorbate) was hyperbolic, giving k_(obs(max)) = 0.41 +- 0.025 min~(-1) and S_(0.5)(H_2O_2) = 1.16 +- 0.18 mM. A kinetic scheme is presented to describe the irreversible inactivation, involving hydroxyl radical generation by reaction of H_2O_2 with Fe(II)-Fe(III) enzyme, reduction of the product Fe(II)-Fe(III) enzyme by ascorbate and reaction of hydroxyl radical with an essential group in the enzyme.