Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.5.1.18 (glutathione S-transferase)
 22,582 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The binding of two different reaction products (p-nitrobenzyl glutathione and the aflatoxin-glutathione conjugate) to mouse glutathione S-transferase A3-3 (mGSTA3-3) has been measured using equilibrium dialysis and a direct fluorescence quenching technique. As expected, p-nitrobenzyl glutathione was found to bind with a stoichiometry of 2.24 +/- 0.17 mol/mol of dimeric enzyme. However, the much larger aflatoxin-glutathione conjugate, 8, 9-dihydro-8-(S-glutathionyl)-9-hydroxyl-aflatoxin B1 (AFB-GSH), was found to bind with a stoichiometry of 1.12 +/- 0.08 mol/mol of dimeric enzyme. p-Nitrobenzyl glutathione bound mGSTA3-3 with a dissociation constant (Kd) of 59 +/- 17 microM while the aflatoxin-glutathione conjugate bound the enzyme with a Kd of 0.86 +/- 0.19 microM. Glutathione competitively inhibited binding of AFB-GSH to mGSTA3-3 with a Ki of 1.5 mM, suggesting that AFB-GSH was binding to the enzyme active site. Although AFB-GSH bound to mGSTA3-3 with a stoichiometry of 1 mol/mol of dimeric enzyme, AFB-GSH completely inhibited activity toward 1-chloro-2, 4-dinitrobenzene, indicating that AFB-GSH binding to one active site alters affinity for 1-chloro-2,4-dinitrobenzene in the active site of the other subunit. To our knowledge, this is the first report of a glutathione S-transferase reaction product which binds to the enzyme with a stoichiometry of 1 mol/mol of dimer.
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PMID:Binding of the aflatoxin-glutathione conjugate to mouse glutathione S-transferase A3-3 is saturated at only one ligand per dimer. 891 Mar 29

A major goal in protein engineering is the tailor-making of enzymes for specified chemical reactions. Successful attempts have frequently been based on directed molecular evolution involving libraries of random mutants in which variants with desired properties were identified. For the engineering of enzymes with novel functions, it would be of great value if the necessary changes of the active site could be predicted and implemented. Such attempts based on the comparison of similar structures with different substrate selectivities have previously met with limited success. However, the present work shows that the knowledge-based redesign restricted to substrate-binding residues in human glutathione transferase A2-2 can introduce high steroid double-bond isomerase activity into the enzyme originally characterized by glutathione peroxidase activity. Both the catalytic center activity (k(cat)) and catalytic efficiency (k(cat)/K(m)) match the values of the naturally evolved glutathione transferase A3-3, the most active steroid isomerase known in human tissues. The substrate selectivity of the mutated glutathione transferase was changed 7000-fold by five point mutations. This example demonstrates the functional plasticity of the glutathione transferase scaffold as well as the potential of rational active-site directed mutagenesis as a complement to DNA shuffling and other stochastic methods for the redesign of proteins with novel functions.
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PMID:Transmutation of human glutathione transferase A2-2 with peroxidase activity into an efficient steroid isomerase. 1202 94