title: Comparative studies on kinetic behavior of horseradish peroxidase isoenzymes.
abstract: Kinetic parameters for each reaction step of the peroxidase-catalyzed reaction were determined by the stopped-flow technique on three distinct isoenzymes: acidic A2, neutral C1, and basic E5. The pH dependence of the reaction for the formation of compound I with hydrogen peroxide was examined. The three isoenzymes had a common ionizing group at about pK 4 which affects the rate constant for the formation of compound I. The heat of ionization determined from the temperature dependence of the dissociation constant of the group strongly suggested that it is of carboxyl nature. The rate constant for isoenzyme A2 was a tenth of those for the other two isoenzymes over the whole range of pH. Furthermore, the thermodynamic parameters of isoenzyme A2 were found to be different from those for the other two isoenzymes. These difference as well as the different behavior in alkaline transition of the isoenzymes are discussed in relation to the sixth ligand of the heme. The rate constant of the reduction of compound I and compound II by ferrocyanide were also determined. In both reduction steps, the pH profiles of the apparent rate constant for isoenzyme A2 and E5 were similar, but they were different from that of C1. The ionization with pK 5.29, which was detected only in isoenzyme C1, may be attributed to a group near the porphyrin ring as a stabilizer for the pi-cation radical.
mesh_terms: D055598:Chemical Phenomena; D002621:Chemistry; D005295:Ferrocyanides; D006735:Horseradish Peroxidase / Q000378:metabolism*; D006861:Hydrogen Peroxide; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D008956:Models, Chemical; D010084:Oxidation-Reduction; D010544:Peroxidases / Q000378:metabolism*; D013816:Thermodynamics
chemical_list: D005295:Ferrocyanides; D007527:Isoenzymes; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase; D010544:Peroxidases