Hiner A N, Hernández-Ruiz J, Rodríguez-López J N, Arnao M B, Varón R, García-Cánovas F, Acosta M
Departamento de Biología Vegetal (Fisiología Vegetal), Universidad de Murcia, Espinardo, Spain.
J Biol Inorg Chem. 2001 Jun;6(5-6):504-16. doi: 10.1007/s007750100219.
The inactivation of horseradish peroxidase A2 (HRP-A2) with H2O2 as the sole substrate has been studied. In incubation experiments it was found that the fall in HRP-A2 activity was non-linearly dependent on H2O2 concentrations and that a maximum level of inactivation of approximately 80% (i.e. approximately 20% residual activity) was obtained with 2,000 or more equivalents of H2O2. Further inactivation was only induced at much higher H2O2 concentrations. Spectral changes during incubations of up to 5 days showed the presence of a compound III-like species whose abundance was correlated to the level of resistance observed. Inactivation was pH dependent, the enzyme being much more sensitive under acid conditions. A partition ratio (r1 approximately equals 1,140 at pH 6.5) between inactivation and catalysis was calculated from the data. The kinetics of inactivation followed single exponential time curves and were H2O2 concentration dependent. The apparent maximum rate constant of inactivation was lambdamax=3.56+/-0.07x10(-4)s(-1) and the H2O2 concentration required to give lambdamax/2 was K2=9.94+/-0.52 mM. The relationship lambdamax<ki has been shown to apply and thus the rate constant of inactivation has been calculated as ki=1.9x10(-3)s(-1). HRP-A2 possessed catalase-like oxygen gas-releasing activity, the catalytic constant being k3=2.2 s(-1), and the affinity for H2O2 as K2=23 mM. Catalase-like activity was pH dependent and favoured under more basic conditions. A mechanistic model has been developed and used to explain the behaviour of HRP-A2. The model suggests that, in common with HRP-C, mechanism-based (suicide) inactivation is being observed but that a fraction of the HRP-A2 is protected from inactivation in the form of a modified compound III species.
以过氧化氢（H₂O₂）作为唯一底物，对辣根过氧化物酶A2（HRP - A2）的失活情况进行了研究。在孵育实验中发现，HRP - A2活性的下降与H₂O₂浓度呈非线性相关，当使用2000或更多当量的H₂O₂时，可获得约80%的最大失活水平（即约20%的残余活性）。只有在更高的H₂O₂浓度下才会进一步诱导失活。长达5天的孵育过程中的光谱变化显示存在一种类似化合物III的物质，其丰度与观察到的抗性水平相关。失活具有pH依赖性，该酶在酸性条件下更为敏感。根据数据计算出失活与催化之间的分配比（在pH 6.5时，r1约等于1140）。失活动力学遵循单指数时间曲线，且与H₂O₂浓度有关。失活的表观最大速率常数为λmax = 3.56 ± 0.07×10⁻⁴ s⁻¹，产生λmax/2所需的H₂O₂浓度为K2 = 9.94 ± 0.52 mM。已证明λmax < ki的关系成立，因此失活速率常数计算为ki = 1.9×10⁻³ s⁻¹。HRP - A2具有类似过氧化氢酶的释放氧气的活性，催化常数为k3 = 2.2 s⁻¹，对H₂O₂的亲和力为K2 = 23 mM。类似过氧化氢酶的活性具有pH依赖性，在更碱性的条件下更有利。已建立并使用一个机理模型来解释HRP - A2的行为。该模型表明，与HRP - C一样，观察到基于机制的（自杀性）失活，但一部分HRP - A2以修饰的化合物III形式受到保护而不被失活。
