A Comparative Study of the Inactivation of Wild‐Type, Recombinant and Two Mutant Horseradish Peroxidase Isoenzymes C by Hydrogen Peroxide and m‐chloroperoxybenzoic Acid

Abstract

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 (m ClO2‐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 m ClO2BzOH 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 m ClO2BzOH 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*. Copyright © 1995, Wiley Blackwell. All rights reserved