Glutathione reductase reaction catalyzed

Figure 45-6. Interaction and synergism between antioxidant systems operating in the lipid phase (membranes) of the cell and the aqueous phase (cytosol). (R-,free radical PUFA-00-, peroxyl free radical of polyunsaturated fatty acid in membrane phospholipid PUFA-OOH, hydroperoxy polyunsaturated fatty acid in membrane phospholipid released as hydroperoxy free fatty acid into cytosol by the action of phospholipase Aj PUFA-OH, hydroxy polyunsaturated fatty acid TocOH, vitamin E (a-tocopherol) TocO, free radical of a-tocopherol Se, selenium GSH, reduced glutathione GS-SG, oxidized glutathione, which is returned to the reduced state after reaction with NADPH catalyzed by glutathione reductase PUFA-H, polyunsaturated fatty acid.)...

Reaction Mechanism and Kinetic Constants for a Reaction Catalyzed by Glutathione Reductase... 

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. 

G6PD is responsible for maintaining adequate levels of NADPH inside the cell. NADPH is a required cofactor in many biosynthetic reactions and also provides electrons to reduce oxidized glutathione (GSSG) to reduced glutathione (GSH) catalyzed by glutathione reductase (Fig. 11-6). 

It is not surprising that enzymes catalyzing such similar chemical reactions should bear striking similarity to one another both structurally and mechanistically. Lipoamide dehydrogenase (34-38), glutathione reductase (39), and thioredoxin reductase (SO, 31) contain, in addition to FAD, a reactive disulfide which is functional in catalysis. These fiavoproteins consist of two identical or near identical polypeptide chains, each with a reactive cystine residue, and two molecules of FAD (31-36). 

Glutathione reductase amino acid composition, 102,104,105 cystine residues, 104 kinetic studies, 138-141 mechanism, 94, 97-98,134 metabolic functions, 129-133 reaction catalyzed, 92 reduction of, 112, 113 specificity of, 92-93 coenzymes and, 94 thiol groups, 141-142 two-electron-reduced enzyme, properties, 133-138... 

A number of enzymes have been characterized that catalyze reactions involving DHA. In addition, other aspects of DHA biochemistry can be deduced from metabolic studies of ascorbic acid. Experiments demonstrating the biological oxidation of AA and reduction of DHA were first made in 1928 (10) and during the next decade several groups studied these reactions. By 1941 Crook (62) was able to separate the ascorbic acid oxidase and DHA reductase activities and to show that glutathione was used in the reductase reaction. 

Spinach leaves contain a dehydroascorbate reductase enzyme that catalyzes Reaction 13 at acidic and neutral pH values, but the enzyme is not, located within the chloroplast (73-76). GSSG produced by Reaction 13 can be re-converted to GSH by glutathione reductase, an enzyme located in the chloroplast (77). 

In brief, the method involves the glutathione reductase-catalyzed reduction of GSSG by NADPH, followed by the reaction of GSH with Ellman s reagent, (5,5 -dithiobis-2-nitrobenzoic acid DTNB). The chromophoric product, TNB, has an absorbance between 405 -12 nm. The reaction can therefore be followed spectrophotometrically (Reaction 3). 

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