Hiner A N, Hernández-Ruíz J, García-Cánovas F, Smith A T, Arnao M B, Acosta M
Departamento de Biología Vegetal (Fisiología Vegetal), Universidad de Murcia, Spain.
Eur J Biochem. 1995 Dec 1;234(2):506-12. doi: 10.1111/j.1432-1033.1995.506_b.x.
The mechanism-based inactivation of four horseradish peroxidase (HRP-C) enzyme variants has been studied kinetically with either hydrogen peroxide or the xenobiotic m-chloroperoxybenzoic acid (mClO2-BzOH) as sole substrate. The concentration and time dependence of inactivation was investigated for the wild-type plant enzyme (HRP-C), the unglycosylated recombinant enzyme (HRP-C*), and two site-directed mutants with Phe143 replaced by Ala ([F143A]HRP-C*) or Arg38 replaced by Lys ([R38K]HRP-C*). The number of turnovers (r) of H2O2 required to completely inactivate the enzymes was found to vary between the different enzymes with HRP-C being most resistant to inactivation (r = 625), HRP-C* and [F143A]HRP-C* being approximately twice as sensitive (r = 335 and 385, respectively) in comparison, and [R38K]HRP-C* being inactivated much more easily (r = 20). In the cases of HRP-C* and [F143A]HRP-C*, compared to HRP-C the differences were due to the absence of glycosylation on the exterior of the proteins, whilst the [R38K]HRP-C* variant exhibited a distinct mechanistic difference. When mClO2BzOH was used as the substrate the differences in sensitivity to inactivation disappeared. The values of r were all around 3 reflecting the strong affinity of mClO2BzOH for the active site. The apparent rate constant for inactivation by H2O2 was found to be about twofold higher in [R38K]HRP-C* than the other enzymes and the catalytic constant for turnover of H2O2 was approximately ten times lower. The affinity of compound I for H2O2 leading to the formation of a transitory intermediate implicated in the inactivation of peroxidase decreased in the order HRP-C, HRP-C*, [F143A]HRP-C*, [R38K]HRP-C*.
以过氧化氢或异生物素间氯过氧苯甲酸（mClO₂ - BzOH）作为唯一底物，对四种辣根过氧化物酶（HRP - C）酶变体基于机制的失活进行了动力学研究。研究了野生型植物酶（HRP - C）、未糖基化重组酶（HRP - C*）以及两个位点定向突变体（Phe143被Ala取代的[F143A]HRP - C和Arg38被Lys取代的[R38K]HRP - C）失活的浓度和时间依赖性。发现完全使酶失活所需的H₂O₂周转数（r）在不同酶之间有所不同，其中HRP - C对失活最具抗性（r = 625），相比之下，HRP - C和[F143A]HRP - C的敏感性约为其两倍（分别为r = 335和385），而[R38K]HRP - C更容易失活（r = 20）。在HRP - C和[F143A]HRP - C的情况下，与HRP - C相比，差异是由于蛋白质外部缺乏糖基化，而[R38K]HRP - C变体表现出明显的机制差异。当使用mClO₂BzOH作为底物时，失活敏感性的差异消失。r值均约为3，反映了mClO₂BzOH对活性位点的强亲和力。发现[R38K]HRP - C中H₂O₂导致失活的表观失活速率常数比其他酶高约两倍，H₂O₂周转的催化常数约低十倍。导致过氧化物酶失活的瞬态中间体形成的化合物I对H₂O₂的亲和力按HRP - C、HRP - C、[F143A]HRP - C*、[R38K]HRP - C*的顺序降低。
