Stepwise binding of nickel to horseradish peroxidase and inhibition of the enzymatic activity.

The incubation of horseradish peroxidase C (HRPC) with millimolar concentrations of nickel, at room temperature and at pH 4.0, induced the progressive formation of a metal-enzyme complex characterized by alterations of the enzyme Soret absorption band that were time- as well as nickel concentration- dependent. For any given incubation period between 1 and 60 min, 2 values for the apparent dissociation constant (K(d)) were found, suggesting the presence of binding sites with different affinities for nickel. The value of each K(d) dropped as the incubation time increased, indicating a progressive stabilization of the metal-enzyme complex. Hill plots suggested a cooperative binding of up to four Ni2+ ions per molecule of HRPC. The inhibition of the enzymatic activity by nickel was studied by following the H2O2-mediated oxidation of o-dianisidine by HRPC under steady-state kinetic conditions. Ni2+ was found to be either a noncompetitive or a mixed inhibitor of HRPC depending both on the duration of preincubation with the enzyme and on Ni2+ concentration. The enzyme remained active only over a limited metal concentration range and data indicated that binding of one Ni2+ affected the substrate binding site, binding of a second Ni2+ affected both substrate and peroxide binding sites, and binding of more than 2 Ni2+ per HRPC molecule led to complete loss of enzymatic activity. Results pointed to the damaging effects of prolonged exposure to heavy metals and also to the existence of a critical metal concentration beyond which immediate abolishing of enzymatic activity was observed.

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 Cd(2+) for various periods of time. The H(2)O(2)-mediated oxidation of o-dianisidine by HRP was used to assess the enzymatic activity. Cd(2+) 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 Cd(2+) concentration. Furthermore, for the same inhibition type, K(i) values dropped as incubation time increased. These results suggested that Cd(2+) would slowly bind to the enzyme and progressively induce conformational changes. Spectrophotometric analysis showed that indeed Cd(2+) altered the heme Soret absorption band on binding HRP and exhibited a K(d) which decreased as the incubation time of HRP with Cd(2+) increased. Hill plots suggested a cooperative binding of up to three Cd(2+) ions per molecule of HRP. Thus, Cd(2+) binding to HRP resulted in progressive inhibition of enzymatic activity with a change in the inhibition type as the number of Cd(2+) 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.