pH‐dependent Properties of a Mutant Horseradish Peroxidase Isoenzyme C in which Arg38 has been Replaced with Lysine

Abstract

Arg38 in the active site of horseradish peroxidase isoenzyme C (HRP-C) has been replaced with lysine by site-directed mutagenesis. As a preclude to a detailed kinetic analysis of this variant, the present study characterizes a pH-dependent cycle of reactions for recombinant horseradish peroxidase isoenzyme C with Arg38 replaced by lysine ([R38K]HRP-C*), which involves time-dependent changes in both specific activity and the electronic absorption spectrum of the enzyme. This pH-dependent cycle resembles that previously suggested for a cytochrome-c peroxidase variant in which Asp235 was replaced with asparagine. When the pH of a solution of resting [R38K]HRP-C* at pH 6.6 (form AH) is raised to pH 8.6, a rapid alkaline transition occurs. This results in spectral changes characteristic of a shift from a predominantly pentacoordinate to a completely hexacoordinate high-spin haem iron (form A-) with a pKa of 7.5. When the pH of a solution of form A- is raised from 8.7 to 12.0, no further spectral changes are observed. The reaction is reversible, but when the high-pH form of the enzyme (A-) is allowed to stand at pH 8.6, it slowly becomes converted into a third enzyme form (form I-) at a rate which is independent of pH (k = 0.56 h-1). When the pH of a sample of form I- is lowered from 8.6 to 6.6, the original low-pH form (AH) of the enzyme is recovered. Recovery of form AH from form I- does not occur via form A-, but via at least one further intermediate, form X. Following a downward pH jump, the rate constant for the formation of form X from form I- shows a small dependence on pH, changing from 48 s-1 at pH 6.8 to 39 s-1 at pH 7.4. The rate of formation of form AH from form X is also pH dependent and biphasic in nature, with measured rate constants ranging from 11.9-2.1 h-1. The possible structures of the different forms of [R38K]HRP-C* are discussed in the light of similar data in the literature for variants of cytochrome-c peroxidase. The properties may be indicative of a greater degree of conformational flexibility within the active site of this mutant caused by the smaller bulk of the lysine side-chain and the probable disruption of a part of the haem-linked hydrogen-bonding network in the distal haem pocket. The wild-type enzyme undergoes no such pH induced changes.