Glutathione reductase substrates

Henderson, G. B., Murgolo, N.J., Kuriyan, J., Osapay, K., Kominos, D., Berry, A., Scrutton, N. S., Hinchlifife, N. W., Perham, R. N. Cerami, A. (1991). Engineering the substrate specificity of glutathione reductase toward that of trypanothione reduction. Proceedings of the National Academy of Sciences USA, 88, 8769-73. 

Perham, R. N., Scrutton, N.S. Berry, A. (1991)- New enzymes for old redesigning the coenzyme and substrate specificities of glutathione reductase. Bioessays, 13, 515-25. 

The reaction catalyzed by the first of these is illustrated in Table 15-2 (reaction type F). The other two enzymes usually promote the reverse type of reaction, the reduction of a disulfide to two SH groups by NADPH (Eq. 15-22). Glutathione reductase splits its substrate into two halves while reduction of the small 12-kDa protein thioredoxin (Box 15-C) simply opens a loop in its peptide chain. The reduction of lipoic acid opens the small disulfide-containing 5-membered ring in that molecule. Each of these flavoproteins also contains within its structure a reducible disulfide group that participates in catalysis. 

The second part of this project will investigate the applicability of the isoenzyme systems Glutathione Reductase and Peptidase A to dried blood analysis. The grantee proposes, also, to incorporate the Gm and Inv allotypes into routine use. Techniques for identifying these genetic markers are well-established for whole blood but must be adapted for dried blood analysis. Persistence studies will be undertaken to determine the viability of these different systems upon drying, to determine the effect of aging, to document the effect of various substrates, and to devise a practical system to type the isoenzymes and allotypes in dried blood. 

Relative to the dithiol DTT but also to other monothiols such as 2-mercaptoeth-anol, GSH is a poor stimulator of microsomal deiodinase activity even when tested in the presence of NADPH and glutathione reductase [52,60,61]. Deiodinase activity of isolated microsomes is supported to a limited extent by GSH if tested with low (nM) but not high (/zM) rT3 concentrations or with T4 as the substrate. This low potency of GSH has led investigators to explore other physiological cofactors. As mentioned above, the paucity of cytoplasmic dihydrolipoamide makes it an unlikely candidate despite its unsurpassed potency [52], This is supported by the finding that addition of NADH, the cofactor for lipoamide hydrogenase, does not stimulate deiodinase activity of kidney homogenates unless supplemented with lipoamide [52]. 

The specificity of these enzymes toward their disulfide substrates is quite remarkable There is virtually no cross-reactivity. Since it is quite difficult to separate glutathione reductase from thioredoxin reductase it... 

The three enzymes are quite specific for their respective pyridine nucleotide substrates. Under conditions normally used for assay, lipoamide dehydrogenase is less than % as active with NADPH as with NADH IS) and thioredoxin reductase is less than 1% as active with NADH as with NADPH 36, Sff). Lipoamide dehydrogenase can transfer electrons to a number of NAD analogs 37). Yeast glutathione reductase is quite specific for NADPH 60), but the erythrocyte enzyme is 20% as active with NADH as with NADPH under the conditions of the standard assay 30,40,61). 

The specificity of glutathione reductase toward its disulfide substrate was emphasized in Section II,A, since there is virtually no reactivity with the substrates of the other pyridine nucleotide-disulfide oxidoreductases. Other authors have emphasized the lack of specificity of this enzyme since it can catalyze the reduction of a variety of mixed disulfides provided that glutathione or y-glutamylcysteine comprises one-half (IP5, 21%) Table IV summarizes these (39, 226-231). It is important to distin-... 

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