A comparative study of the inactivation of wild-type, recombinant and two mutant horseradish peroxidase isoenzymes C by hydrogen peroxide and m-chloroperoxybenzoic acid.
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*.
Hiner
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AN
Departamento de Biología Vegetal (Fisiología Vegetal), Universidad de Murcia, Spain.
Hernández-Ruíz
J
J
García-Cánovas
F
F
Smith
A T
AT
Arnao
M B
MB
Acosta
M
M
D001665:Binding Sites; D002723:Chlorobenzoates / Q000494:pharmacology*; D006735:Horseradish Peroxidase / Q000187:drug effects* / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D007527:Isoenzymes / Q000187:drug effects* / Q000378:metabolism; D009154:Mutation; D011994:Recombinant Proteins / Q000187:drug effects
D002723:Chlorobenzoates; D007527:Isoenzymes; D011994:Recombinant Proteins; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase; C000433:3-chloroperbenzoic acid