Irreversible inactivation of purple acid phosphatase by hydrogen peroxide and ascorbate.
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 H2O2 to a much smaller extent than the Cu(II)-Fe(III) derivative, whereas the Zn(II)-Fe(III) derivative was stable to H2O2 treatment. The rates of inactivation of the Cu(II)-Fe(III) and Fe(II)-Fe(III) enzymes in the presence of H2O2 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 H2O2 in the presence of ascorbate obeyed pseudo first-order kinetics. A plot of kobs for this reaction against H2O2 concentration (at saturating ascorbate) was hyperbolic, giving kobs(max) = 0.41 +/- 0.025 min-1 and S0.5(H2O2) = 1.16 +/- 0.18 mM. A kinetic scheme is presented to describe the irreversible inactivation, involving hydroxyl radical generation by reaction of H2O2 with Fe(II)-Fe(III) enzyme, reduction of the product Fe(III)-Fe(III) enzyme by ascorbate and reaction of hydroxyl radical with an essential group in the enzyme.
Beck
J L
JL
Department of Biochemistry, University of Queensland, St Lucia, Australia.
Durack
M C
MC
Hamilton
S E
SE
de Jersey
J
J
D000135:Acid Phosphatase / Q000037:antagonists & inhibitors*; D001205:Ascorbic Acid / Q000494:pharmacology*; D003198:Computer Simulation; D018360:Crystallography, X-Ray; D006023:Glycoproteins / Q000037:antagonists & inhibitors*; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D010940:Plant Proteins / Q000037:antagonists & inhibitors*; D011487:Protein Conformation; D013054:Spectrophotometry, Atomic
D006023:Glycoproteins; D010940:Plant Proteins; D006861:Hydrogen Peroxide; C091137:purple acid phosphatase; D000135:Acid Phosphatase; D001205:Ascorbic Acid