Beck J L, Durack M C, Hamilton S E, de Jersey J
Department of Biochemistry, University of Queensland, St Lucia, Australia.
J Inorg Biochem. 1999 Apr;73(4):245-52. doi: 10.1016/s0162-0134(99)00022-7.
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.
用过氧化氢处理猪尿囊液酸性磷酸酶的Cu(II)-Fe(III)衍生物会导致该酶不可逆失活，并使可见吸收光谱强度损失一半。竞争性抑制剂磷酸盐可防止这种失活，这表明失活是由于活性位点处的反应所致。天然的Fe(II)-Fe(III)酶被H2O2不可逆失活的程度比Cu(II)-Fe(III)衍生物小得多，而Zn(II)-Fe(III)衍生物对H2O2处理稳定。在H2O2存在下，添加抗坏血酸会增加Cu(II)-Fe(III)和Fe(II)-Fe(III)酶的失活速率。这些结果表明涉及芬顿型反应，产生与必需活性位点基团反应的羟基自由基。对Fe(II)-Fe(III)酶进行的实验表明，在抗坏血酸存在下，H2O2导致的不可逆失活符合准一级动力学。该反应的表观一级速率常数kobs对H2O2浓度（在抗坏血酸饱和时）的作图呈双曲线，得到kobs(max)=0.41±0.025 min-1和S0.5(H2O2)=1.16±0.18 mM。提出了一个动力学方案来描述不可逆失活，包括H2O2与Fe(II)-Fe(III)酶反应产生羟基自由基、抗坏血酸还原产物Fe(III)-Fe(III)酶以及羟基自由基与酶中的必需基团反应。
