A COMPARATIVE-STUDY OF THE INACTIVATION OF WILD-TYPE, RECOMBINANT AND2 MUTANT HORSERADISH-PEROXIDASE ISOENZYMES-C BY HYDROGEN-PEROXIDE ANDM-CHLOROPEROXYBENZOIC ACID

The mechanism-based inactivation of four horseradish peroxidase (HRP-C ) enzyme variants has been studied kinetically with either hydrogen pe roxide or the xenobiotic m-chloroperoxybenzoic acid (mClO(2)-BzOH) as sole substrate. The concentration and time dependence of inactivation was investigated for the wild-type plant enzyme (HRP-C), the unglycosy lated 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 i nactivate 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*, compar ed to HRP-C the differences were due to the absence of glycosylation o n the exterior of the proteins, whilst the [R38K]HRP-C variant exhibi ted a distinct mechanistic difference, When mClO(2)BzOH was used as th e substrate the differences in sensitivity to inactivation disappeared . The values of r were all around 3 reflecting the strong affinity of mClO(2)BzOH for the active site, The apparent rate constant for inacti vation by H2O2 was found to be about twofold higher in [R38K]HRP-C th an the other enzymes and the catalytic constant for turnover of H2O2 w as approximately ten times lower, The affinity of compound I for H2O2 leading to the formation of a transitory intermediate implicated in th e inactivation of peroxidase decreased in the order HRP-C, HRP-C, [F1 43A]HRP-C, [R38K]HRP-C*.