Studies on Horseradish Peroxidase

The kinetics of the oxidation of p-cresol by Compound II of horseradish peroxidase has been studied by the stopped flow technique at an ionic strength of 0.11 from pH 2 to 11. In acid solution the reaction is kinetically first order in jcresol, but in the alkaline region a saturation effect attributable to complex formation is observed. At very high pH an additional second order reaction between p-cresol and the complex is detectable. The existence of the complex is confirmed by spectrophotometric determination of its dissociation constant. Since this constant and the over-all second order rate constant do not depend on any common ionizing group in the enzyme, it is argued that the complex is unproductive in nature. Use of the diffusion-controlled limit shows that p-cresol reacts in the unionized form over the experimental pH range, and that electrostatic interactions are therefore not responsible for the degree of complexity of the pH log rate profile.