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.
Keyhani
Jacqueline
J
Laboratory for Life Sciences, Sarve Sharghi 34, 19979 Tehran, Iran. keyhanie@ibb.ut.ac.ir
Keyhani
Ezzatollah
E
Zarchipour
Sekineh
S
Tayefi-Nasrabadi
Hossein
H
Einollahi
Nahid
N
D002384:Catalysis; D006735:Horseradish Peroxidase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007700:Kinetics; D009532:Nickel / Q000378:metabolism*; D011485:Protein Binding
D009532:Nickel; D006735:Horseradish Peroxidase