Glutathione reductase, complex with

Cohen, M.D., A.C. Sen, and C.-I. Wei. 1987. Ammonium metavanadate complexation with glutathione disulfide A contribution to the inhibition of glutathione reductase. Inorg. Chim. Acta 138 91-93. 

The stability of EH2 is very species dependent. All of the above results refer to the pig heart enzyme and, where tested, to other mammalian species. It was initially reported that no long wavelength absorption was observed upon reduction of E. coli enzyme with NADH 109), but reduction by 1 equivalent of NADH or dihydrolipoamide leads to the formation of 25% of the maximal 2-electron-reduced species 108) and similar results are obtained with the Azotobacter enzyme 114)- That this species is the catalytically important one in the E. coli enzyme as well as in the mammalian enzyme has also been demonstrated 50). Reduction with dihydrolipoamide in the rapid reaction spectrophotometer at 2° results in the full formation of EH2 followed by the slow k = 13 min, 1 mAf dihydrolipoamide) four-electron reduction. The spectrum of EHa generated in this way is shown in Fig. 7 and is identical with that of the pig heart enzyme. The 2-electron-reduced form, EHj of lipoamide dehydrogenase of spinach 99) may be somewhat unstable however, spectrally it is difficult to distinguish between instability and formation of the EHa-NADH complex (see above) on the basis of available spectral data. Either phenomenon could lead to inhibition by excess NADH. In glutathione reductase it is possible that the complex can be rapidly reoxidized by glutathione 53). 

The reactions in Eq. (10), proceeding to the right from E, are part of normal catalysis as shown in Fig. 10 (SI, S3). The association of E with NADP leads to a dead end complex. In the reaction of yeast glutathione reductase with NADPH, EH,-NADPH appears to be formed in the dead time of the rapid reaction spectrophotometer (co. 3 msec) when observation is at 540 nm (344), however, if 3.4 /iM EH,(free) is mixed with 20 fiM NADPH, Eq. (11), a minimum rate of complex formation of... 

Kinetic parameters are given in Table VI. Plots of 1/v against 1/(S) ve parallel lines both at 4° and 25° on this basis the assumption has been made that a binary complex mechanism is operative. As with lipo-amide dehydrogenase and glutathione reductase, this assumption is com-... 

Fia. 11. Complex formation between the 2-electron-reduced (EHj) form of E. coli glutathione reductase and NADPH. EHz was produced by anaerobic reduction with borohydride time was allowed for the slight excess of borohydride to react with water before begitming the titration with NADPH. 1, Oxidized 2, EHa 3, EHa-1-0.45 equivalent NADPH 4, EHa-f 0.90 equivalent NADPH and 5, EHa -)- 2.75 equivalents NADPH. 

The early kinetic studies on glutathione reductase did not include investigation of product inhibition, so vital to a proper interpretation of kinetic data in the elucidation of the mechanism 2 7, 2 8). In the one case where product inhibition patterns were observed, they were not interpreted by more recent kinetic theory (40). Subsequent kinetic analyses see below), in which product inhibition patterns have been obtained, were either completed prior to the discovery of the EH2-NADPH complex 53) or have not considered it. Furthermore, the product inhibition patterns have been carried out at only a single level of the fixed substrate it is essential that the patterns be obtained at more than one level of fixed substrate, especially where dead end complexes are involved 249) as has been so amply demonstrated with lipoamide dehydrogenase 95, 157). In spite of these deficiencies, the more recent kinetic studies have yielded much useful information. 

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