Studies on Horseradish Peroxidase

Abstract Binding of p-crestol to native horseradish peroxidase was investigated by differential spectrophotometry, and the value 103 Kdiss = 3 m was obtained at neutral and acid pH; binding is not competitive with that of cyanide and hydroxide. The Soret region spectrum of Compound II of the enzyme was measured in the steady state at pH 4.26, 6.89, and 10.95, and the differences were found to be too small to be attributed to the acid dissociations of an iron-bound group. The kinetics of the reactions between Compound II and p-cresol, ferrocyanide, and iodide was investigated in 94% D2O. Almost no solvent isotope effect was found on the ionization of the group of pKa 8.6. The reaction with p-cresol gave kd/kh = 0.45 ± 0.04, which was attributed to a rate-determining proton transfer. In regions of the pH log rate profile having a slope of - 1 ferrocyanide gave kd/kh = 2.6 ± 0.3 at high pH and 2.9 ± 0.6 at low pH, whereas iodide gave kd/kh = 4.0 ± 0.4. The pH rate profiles, isotope effects, and over-all rates of the three reactions were correlated in terms of a mechanism involving intramolecular general acid catalysis. In sufficiently acidic solutions this ultimately gives way to a specific acid-catalyzed mechanism, and the mechanistic changeover occurs at a higher pH the more difficult the over-all reaction.