Heterogeneous inhibition of horseradish peroxidase activity by cadmium

Inhibition of horseradish peroxidase (HRP) activity by cadmium was studied under steady-state kinetic conditions after preincubation of the enzyme with millimolar concentrations of Cd2+ for various periods of time. The H2O2-mediated oxidation of o-dianisidine by HRP was used to assess the enzymatic activity. Cd2+ was found to be either a noncompetitive inhibitor of HRP or a mixed inhibitor of HRP depending both on the duration of incubation with HRP and on Cd2+ concentration. Furthermore, for the same inhibition type, K-i values dropped as incubation time increased. These results suggested that Cd2+ would slowly bind to the enzyme and progressively induce conformational changes. Spectrophotometric analysis showed that indeed Cd2+ altered the heme Soret absorption band on binding HRP and exhibited a K-d which decreased as the incubation time of HRP with Cd2+ increased. Hill plots suggested a cooperative binding of up to three Cd2+ ions per molecule of HRP. Thus, Cd2+ binding to HRP resulted in progressive inhibition of enzymatic activity with a change in the inhibition type as the number of Cd2+ ions per HRP molecule increased. Results also illustrated the potential danger of long-term exposure to heavy metals, even for enzymes with low affinity for them. (C) 2003 Elsevier Science B.V. All rights reserved.