Inactivation-reactivation of two-electron reduced Escherichia coli glutathione reductase involving a dimer-monomer equilibrium.
Glutathione reductase from Escherichia coli is inactivated when incubated with either NADPH or NADH. The process is inversely dependent on the enzyme concentration. Inactivation is rapid and monophasic with 1 microM NADPH and 1 nM enzyme FAD giving a t1/2 of 1 min. Complex formation between NADPH and the two-electron reduced enzyme (EH2) at higher levels of NADPH protects against rapid inactivation. NADP+, produced in a side reaction with oxygen, also protects by forming a complex with EH2. These complexes make analysis of the concentration dependence of the inactivation process difficult. Inactivation with NADH, where complexes do not interfere, is slower but can be analyzed more readily. With 152 microM NADH and 5.4 nM enzyme FAD, the time required for 50% inactivation is 17 min. The process is markedly biphasic, reaching the final inactivation level after 5-7 h. Analysis of the relationship between the final level of inactivation with NADH and the enzyme concentration indicates that inactivation is due to dissociation of the normally dimeric enzyme. Thus, the position of the dimer-monomer equilibrium between an active dimeric two-electron reduced species and an inactive monomeric two-electron reduced form determines the enzyme activity. An apparent equilibrium constant (Kd) for dissociation of dimer obtained from the anaerobic concentration dependent inactivation curves is 220 nM. Enzyme inactivated with NADH can be reactivated with glutathione, and the reactivation kinetics are second order, monomer-monomer over 75% of the reaction with an average apparent association rate constant (ka) of 13.1 (+/- 5.5) X 10(6) M-1 min-1.
Arscott
L D
LD
Veterans Administration Medical Center, Ann Arbor, Michigan.
Drake
D M
DM
Williams
C H
CH
D004793:Enzyme Reactivators; D004926:Escherichia coli / Q000201:enzymology*; D005978:Glutathione / Q000494:pharmacology; D005980:Glutathione Reductase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009243:NAD / Q000494:pharmacology; D009249:NADP / Q000494:pharmacology; D010084:Oxidation-Reduction; D011487:Protein Conformation
D004793:Enzyme Reactivators; D009243:NAD; D009249:NADP; D005980:Glutathione Reductase; D005978:Glutathione