Open AccessA Comparative Study of the Inactivation of Wild‐Type, Recombinant and Two Mutant Horseradish Peroxidase Isoenzymes C by Hydrogen Peroxide and m ‐chloroperoxybenzoic Acid
Author(s) -
Hiner Alexander N. P.,
HernándezRuíz Josefa,
GarcíaCánovas Francisco,
Smith Andrew T.,
Arnao Marino B.,
Acosta Manuel
Publication year - 1995
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1995.506_b.x
Subject(s) - horseradish peroxidase , hydrogen peroxide , chemistry , peroxidase , enzyme , substrate (aquarium) , mutant , biochemistry , recombinant dna , glycosylation , enzyme kinetics , stereochemistry , peroxide , active site , biology , organic chemistry , ecology , gene
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 ClO 2 ‐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 H 2 O 2 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 ClO 2 BzOH 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 ClO 2 BzOH for the active site. The apparent rate constant for inactivation by H 2 O 2 was found to be about twofold higher in [R38K]HRP‐C* than the other enzymes and the catalytic constant for turnover of H 2 O 2 was approximately ten times lower. The affinity of compound I for H 2 O 2 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*.