Kinetic analysis of the acid-alkaline conversion of horseradish peroxidases

Abstract
The nature of the acid-alkaline conversion of horseradish peroxidases was studied by measuring 4 rate constants in reactions, E + H+ (k1) .dblarw. (k2) EH+ and E + H2O (k3) .dblarw. (K4) EH+ + OH-, where EH+ and E denote the acid and alkaline forms of the enzymes. The values of k1, (k2 + k3), and k4 were obtained by measuring the relaxation rates of the acid .fwdarw. alkaline and alkaline .fwdarw. acid conversions by means of the pH jump method with a stopped-flow apparatus. The value of k3 was also obtained by measuring the rate of reactions between hydrogen peroxide and peroxidases at alkaline pH. The measurements were conducted with 4 peroxidases having different pKa values: peroxidase A (pKa = 9.3), peroxidase C (pKa = 11.1), diacetyldeuteroperoxidase A (pKa 7.7) and diacetyldeuteroperoxidase C (pKa = 9.1). The value of k1 was about 1010 M-1 s-1 in the reaction of the 4 enzymes while k4 was quite different between the enzymes. The pKa was determined by k3 and k4 for the natural peroxidases and by k1 and k2 for the diacetyldeuteroperoxidases. The mechanisms of the acid-alkaline conversion was discussed in comparison with that of metmyoglobin [mammalian].