Human erythrocyte glutathione reductase: pH dependence of kinetic parameters.
Human erythrocyte glutathione reductase catalyzes the pyridine nucleotide dependent reduction of oxidized glutathione (GSSG). The pH dependence of the kinetic parameters V and V/K for three reduced pyridine nucleotide substrates, the Ki's for three competitive inhibitors (versus NADPH), and the temperature dependence of the V pH profile have been determined. Below pH 8, V and V/K for NADPH, 2',3'-cyclic-NADPH, and NADH are pH independent. In the basic pH region, both V and V/K for the three substrates are pH dependent. All three of the V profiles decrease with increasing pH as a group with a pKa of approximately 9.2 is titrated. The V/K profiles for NADPH, 2',3'-cyclic-NADPH, and NADH decrease at high pH as a group with a pKa of greater than 9.8, 8.9, and 8.8, respectively, is deprotonated. The Ki's for ATP-ribose and 2',5'-ADP are pH independent below pH 8 but increase in the basic region as a group with a pKa of about 8.8 and 8.5, respectively, is deprotonated. The Ki of AADP is pH independent between pH 6 and 9. These studies suggest that binding interactions between the 2'-phosphate of NADPH and the enzyme are predominately nonionic. The temperature dependence of the pK observed in all V pH profiles allows the calculation of an enthalpy of ionization of 3.2 kcal/mol for this group. The high pK and low enthalpy of ionization suggest that the protonation state of the His-467'-Glu-472' ion pair observed in the structure of human erythrocyte glutathione reductase influences proton-transfer steps occurring in the oxidative half-reaction.
Wong
K K
KK
Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461.
Blanchard
J S
JS
D001665:Binding Sites; D001667:Binding, Competitive; D004912:Erythrocytes / Q000201:enzymology*; D005980:Glutathione Reductase / Q000037:antagonists & inhibitors / Q000097:blood*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D009249:NADP / Q000378:metabolism; D013696:Temperature
D009249:NADP; D005980:Glutathione Reductase