Effect of thiocyanate on the peroxidase and pseudocatalase activities of Leishmania major ascorbate peroxidase.
We report here that the Leishmania major ascorbate peroxidase (LmAPX), having similarity with plant ascorbate peroxidase, catalyzes the oxidation of suboptimal concentration of ascorbate to monodehydroascorbate (MDA) at physiological pH in the presence of added H(2)O(2) with concurrent evolution of O(2). This pseudocatalatic degradation of H(2)O(2) to O(2) is solely dependent on ascorbate and is blocked by a spin trap, alpha-phenyl-n-tert-butyl nitrone (PBN), indicating the involvement of free radical species in the reaction process. LmAPX thus appears to catalyze ascorbate oxidation by its peroxidase activity, first generating MDA and H(2)O with subsequent regeneration of ascorbate by the reduction of MDA with H(2)O(2) evolving O(2) through the intermediate formation of O(2)(-). Interestingly, both peroxidase and ascorbate-dependent pseudocatalatic activity of LmAPX are reversibly inhibited by SCN(-) in a concentration dependent manner. Spectral studies indicate that ascorbate cannot reduce LmAPX compound II to the native enzyme in presence of SCN(-). Further kinetic studies indicate that SCN(-) itself is not oxidized by LmAPX but inhibits both ascorbate and guaiacol oxidation, which suggests that SCN(-) blocks initial peroxidase activity with ascorbate rather than subsequent nonenzymatic pseudocatalatic degradation of H(2)O(2) to O(2). Binding studies by optical difference spectroscopy indicate that SCN(-) binds LmAPX (Kd = 100 +/- 10 mM) near the heme edge. Thus, unlike mammalian peroxidases, SCN(-) acts as an inhibitor for Leishmania peroxidase to block ascorbate oxidation and subsequent pseudocatalase activity.
Dolai
Subhankar
S
Division of Structural Biology and Bio-informatics, Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Kolkata-700 032, India.
Yadav
Rajesh K
RK
Datta
Alok K
AK
Adak
Subrata
S
D000818:Animals; D060387:Ascorbate Peroxidases; D001205:Ascorbic Acid / Q000378:metabolism; D002374:Catalase / Q000378:metabolism*; D003001:Cloning, Molecular; D006139:Guaiacol / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism; D007700:Kinetics; D018320:Leishmania major / Q000201:enzymology*; D008024:Ligands; D010084:Oxidation-Reduction; D010100:Oxygen / Q000032:analysis; D010544:Peroxidases / Q000378:metabolism*; D015800:Protozoan Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013861:Thiocyanates / Q000494:pharmacology*
D008024:Ligands; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D013861:Thiocyanates; D006139:Guaiacol; D006861:Hydrogen Peroxide; D010544:Peroxidases; C038109:pseudocatalase; D060387:Ascorbate Peroxidases; D002374:Catalase; C031760:thiocyanate; D001205:Ascorbic Acid; D010100:Oxygen