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TAILIEUCHUNG - The mechanism of inhibition of human erythrocyte catalase by azide

In order to elucidate the kinetic mechanism of human erythrocyte catalase a well-known substrate, H2O2 and inhibitor azide were used. The kinetics conformed to the Michaelis-Menten model. Lineweaver-Burk plots for H2O2 at different fixed concentrations of azide were linear and intersected on the abscissa indicating a noncompetitive or irreversible type of inhibition. | Turk J Biol 28 (2004) 65-70 © TÜB‹TAK The Mechanism of Inhibition of Human Erythrocyte Catalase by Azide Yasemin AKSOY, Mevhibe BALK, ‹. Hamdi Ö⁄Üfi, Nazmi ÖZER Department of Biochemistry, Faculty of Medicine, Hacettepe University, 06100 Ankara - TURKEY Received: Abstract: In order to elucidate the kinetic mechanism of human erythrocyte catalase a well-known substrate, H2O2 and inhibitor azide were used. The catalase-mediated conversion of H2O2 to H2O and O2, in the presence and absence of azide, was studied in 50 mM phosphate buffer pH at 37 ºC under conditions where the peroxidation side reaction (RH2 + H2O2 → R + 2H2O) is negligible ([H2O2] ≤ 25 mM; assay time, 10 s). The kinetics conformed to the Michaelis-Menten model. Lineweaver-Burk plots for H2O2 at different fixed concentrations of azide were linear and intersected on the abscissa indicating a noncompetitive or irreversible type of inhibition. To identify the inhibition type Vm vs. [E] plots were constructed at different [N3-]. The plots were linear and converged at the origin, indicating that N3- is a noncompetitive inhibitor. Using a non-linear curve-fitting program, the kinetic parameters were calculated. The Km value for H2O2, and the Ki value for azide were found to be ± mM and ± µM, respectively. Key Words: Human erythrocytes, catalase, substrate kinetics, azide inhibition ‹nsan Eritrosit Katalaz›n›n Azid ‹nhibisyonunun Mekanizmas› Özet: ‹nsan alyuvar katalazının kinetik mekanizmasını aydınlatabilmek için iyi bilinen substratı, H2O2 ve inhibitörü azid kullanıldı. Katalaz aracılı¤ıyla azid varlı¤ında ve yoklu¤unda hidrojen peroksitin su ve oksijene dönüflümü, 50 mM potasyum fosfat tamponunda, pH ve 37 ºC’de peroksidasyon yan reaksiyonunun (RH2 + H2O2 → R + 2H2O ) ihmal edildi¤i koflullarda gerçeklefltirildi ([ H2O2 ] ≤ 25 mM; reaksiyon süresi, 10 sn). Kineti¤in Michaelis-Menten modeline uydu¤u saptandı. Sabit azid ve de¤iflken H2O2 deriflimlerinde çizilen

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