Glutathione catalysis and the reaction mechanisms of glutathione-dependent enzymes.

Publisher Summary The chapter discusses the biologically relevant chemistry of glutathione (GSH) and its occurrence in microbial cells The GSH-related biochemical reactions and the physiological roles of GSH are summarized The biosynthesis of GSH is remarkable in two ways: it is mRNA independent, and the glutamic residue is joined in an unusual peptide linkage of the γ -carbon atom to the cysteine residue Due to this structural peculiarity, GSH is protected against proteolytic cleavage The GSH status of cells is defined by the total concentration of GSH and the nature and distribution of the possible forms of occurrence of the tripeptide in the cell GSH and related compounds are widespread in the microbial world, especially amongst organisms with an aerobic lifestyle This observation emphasizes the role of GSH in cellular protection against by-products generated by oxidative metabolism, but it does not limit its functions to this role Glutathione acts as an enzyme cofactor, transport component, nucleophilic substrate, and sulphur reservoir; and participates in key cellular processes such as protein synthesis and degradation, regulation of enzyme activity, synthesis of DNA, and maintenance of the integrity of cell membranes and organelles Having a functional diversity, GSH is interrelated with a number of metabolic pathways and its intracellular modulation could have an impact on the entire cell, making it extremely difficult to associate directly a given cellular end-point with one molecule or system Both in Escherichia coli and Saccharomyces cerevisiae, GSH plays an important role in cellular protection during chemical stresses in spite of the fact that key enzymes of detoxification, such as GSH peroxidase and GSH S-transferase, remain at a low level

Metabolism and functions of glutathione in micro-organisms.

The structural differences between trypanothione reductase of Trypanosoma cruzi and human glutathione reductase, an enzyme of known three-dimensional structure, offer an opportunity for rational drug design against Chagas' disease. As a first step in the analysis of the parasite enzyme we report its purification and characterization. 2.2 mg trypanothione reductase was extracted from 33 g wet weight of cultured epimastigotes or from 4 g lyophilized cells. The flavoenzyme was purified 2400-fold to homogeneity in three steps with an overall yield of 45%. The enzyme is a dimer with a subunit Mr of 50,000. Using NADPH (Km = 5 microM) and trypanothione disulfide (Km = 45 microM) as substrates, a turnover number of 14,200 min-1 was estimated. Trypanothione reductase, the parasite enzyme, and glutathione reductase, the host enzyme, exhibit mutually exclusive specificities for their respective disulfide substrates. When screening cell cultures or column eluates for the presence of trypanothione reductase, a microassay based on Ellman's reagent as indicator was used. A mixture of regioisomeric glutathionylspermidine disulfides isolated from Escherichia coli served as substrate in this microassay. Experimentally, the catalytic cycle of the enzyme can be subdivided into the half-reactions Eox + NADPH + H+----EH2 + NADP+, and EH2 + trypanothione disulfide----Eox + dihydrotrypanothione. This is also true for the crystallized enzyme in the presence of 2 M (NH4)2SO4. The spectral properties of trypanothione reductase both in the oxidized form (Eox) and in the two-electron-reduced form (EH2) closely resemble those of human glutathione reductase. Both proteins contain a flavin and a redox-active disulfide at the catalytic site. After reduction of Eox to EH2, trypanothione reductase can be inactivated by specifically alkylating one of the nascent active-site thiols.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1987.tb11002.x

Trypanothione reductase from Trypanosoma cruzi. Purification and characterization of the crystalline enzyme.

Evolution of glutathione metabolism.

Thioredoxin Reductase-2 Is Essential for Keeping Low Levels of H2O2 Emission from Isolated Heart Mitochondria

The disulfide reducing activities of GSSG-and CoASSG-reductases were measured on partially purified extracts from a variety of prokaryotes and eukaryotes.

R. N. Ondarza

Papers

Glutathione reductase in evolution.