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An Essential Role of Active Site Arginine Residue in Iodide Binding and Histidine Residue in Electron Transfer for Iodide Oxidation by Horseradish peroxidase

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Title An Essential Role of Active Site Arginine Residue in
Iodide Binding and Histidine Residue in Electron
Transfer for Iodide Oxidation by Horseradish
peroxidase
 
Creator Adak, Subrata
Bandyopadhyay, Debashis
Bandyopadhyay, Uday
Banerjee, Ranajit K
 
Subject Structural Biology & Bioinformatics
 
Description The objective of the present study is to delineate the role of active site arginine and histidine residues of horseradish peroxidase
(HRP) in controlling iodide oxidation using chemical modification technique. The arginine specific reagent, phenylglyoxal
(PGO) irreversibly blocks iodide oxidation following pseudofirst order kinetics with second order rate constant of 25.12 min–1
M–1. Radiolabelled PGO incorporation studies indicate an essential role of a single arginine residue in enzyme inactivation.
The enzyme can be protected both by iodide and an aromatic donor such as guaiacol. Moreover, guaiacol-protected enzyme
can oxidise iodide and iodide-protected enzyme can oxidise guaiacol suggesting the regulatory role of the same active site
arginine residue in both iodide and guaiacol binding. The protection constant (Kp) for iodide and guaiacol are 500 and 10 µM
respectively indicating higher affinity of guaiacol than iodide at this site. Donor binding studies indicate that guaiacol
competitively inhibits iodide binding suggesting their interaction at the same binding site. Arginine-modified enzyme shows
significant loss of iodide binding as shown by increased Kd value to 571 mM from the native enzyme (Kd = 150 mM). Although
arginine-modified enzyme reacts with H2O2 to form compound II presumably at a slow rate, the latter is not reduced by iodide
presumably due to low affinity binding.
The role of the active site histidine residue in iodide oxidation was also studied after disubstitution reaction of the histidine
imidazole nitrogens with diethylpyrocarbonate (DEPC), a histidine specific reagent. DEPC blocks iodide oxidation following
pseudofirst order kinetics with second order rate constant of 0.66 min–1 M–1. Both the nitrogens (d, e) of histidine imidazole
were modified as evidenced by the characteristic peak at 222 nm. The enzyme is not protected by iodide suggesting that
imidazolium ion is not involved in iodide binding. Moreover, DEPC-modified enzyme binds iodide similar to the native enzyme.
However, the modified enzyme does not form compound II but forms compound I only with higher concentration of H2O2
suggesting the catalytic role of this histidine in the formation and autoreduction of compound I. Interestingly, compound I thus formed is not reduced by iodide indicating block of electron transport from the donor to the compound I. We suggest that an active site arginine residue regulates iodide binding while the histidine residue controls the electron transfer to the heme ferryl
group during oxidation.
 
Date 2001
 
Type Article
PeerReviewed
 
Format application/pdf
 
Identifier http://www.eprints.iicb.res.in/877/1/MOLECULAR_AND_CELLULAR_BIOCHEMISTRY%2C_218(_1%2D2)%2C_1%2D11%2C2001[63].pdf
Adak, Subrata and Bandyopadhyay, Debashis and Bandyopadhyay, Uday and Banerjee, Ranajit K (2001) An Essential Role of Active Site Arginine Residue in Iodide Binding and Histidine Residue in Electron Transfer for Iodide Oxidation by Horseradish peroxidase. Molecular and Cellular Biochemistry, 218 (1-2). pp. 1-11.
 
Relation http://dx.doi.org/10.1023/A:1007154515475
http://www.eprints.iicb.res.in/877/