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On the kinetics of human erythrocyte glutathione disulfide reductase: Does the enzyme really play ping pong

Human erythrocyte glutathione disulfide reductase studied at 37˚C in 100 mM potassium phosphate buffer, pH 7.4, is subject to partial inhibition by GSSG at low [NADPH]. Saturation by NADPH is hyperbolic at low [GSSG] and becomes increasingly sigmoidal at high [GSSG]. NADP+ is a competitive inhibitor with respect to NADPH at 20-1000 µM GSSG and an uncompetitive inhibitor with respect to GSSG at 33-120 µM NADPH. | Tr. J. of Biology 23 (1999) 143-151 © TÜBİTAK On The Kinetics of Human Erythrocyte Glutathione Disulfide Reductase: Does The Enzyme Really Play ‘Ping-Pong’? Hamdi ÖGÜS, Nazmi ÖZER Department of Biochemistry, Faculty of Medicine, Hacettepe University, 06100 Ankara-TURKEY Received: 20.11.1997 Abstract:: Human erythrocyte glutathione disulfide reductase studied at 37˚C in 100 mM potassium phosphate buffer, pH 7.4, is subject to partial inhibition by GSSG at low [NADPH]. Saturation by NADPH is hyperbolic at low [GSSG] and becomes increasingly sigmoidal at high [GSSG]. NADP+ is a competitive inhibitor with respect to NADPH at 20-1000 µM GSSG and an uncompetitive inhibitor with respect to GSSG at 33-120 µM NADPH. Studied under special conditions (10 µM NADPH and 1000 µM GSSG), NADP+ exhibits a two-fold effect, inhibition being preceded by activation in the 050 µM effector concentration range. The data are consistent with the ping-pong / sequential ordered hybrid model diverging at the level of E.NADPH. A composite sequential ordered mechanism is suggested, involving two conformational states of free enzyme at equilibrium. While one state is selective for NADPH as the first substrate, the second state adds GSSG to initiate the catalytic cycle. The pathway proceeding through E.GSSG has an apparently lower turnover number than that proceeding through E.NADPH. Key Words: Glutathione disulfide reductase, kinetics, mechanism, NADP+ inhibition, human erythrocytes. İnsan Eritrosit Glutatyon Disülfit Reduktazının Kinetiği Üzerine Enzim Gerçekten ‘Ping-Pong’ Mekanizma mı Gösterir? Özet: İnsan eritrositi glutatyon disülfit redüktazı, 100 mM potasyum fosfat (pH 7.4, 37˚C) tamponunda çalışıldığında, düşük [NADPH] derişiminde GSSG’nin kısmi inhibisyon yaptığı bulundu. Düşük GSSG derişimlerinde, NADPH doygunluk grafiğinin hiperbolik olduğu ancak GSSG derişimi arttıkça sigmoidisitenin de arttığı gözlendi. NADP+, 20-1000 µM GSSG derişimlerinde, NADPH’la kompetitif; 33-120 µM NADPH .