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Engineering of an intersubunit disulphide bridge in glutathione reductase from Escherichia coli
Author(s) -
Scrutton Nigel S.,
Berry Alan,
Perham Richard N.
Publication year - 1988
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(88)81028-7
Subject(s) - escherichia coli , glutathione reductase , chemistry , bridge (graph theory) , glutathione , biochemistry , protein engineering , enzyme , stereochemistry , biology , glutathione peroxidase , gene , anatomy
By site‐directed mutagenesis, Thr‐75 was converted to Cys‐75 in the glutathione reductase (EC 1.6.4.2 of Escherichia coli . This led to the spontaneous formation of an intersubunit disulphide bridge across the 2‐fold axis of the dimeric enzyme. The disulphide bridge had no deleterious effect on the catalytic activity, but nor did it increase the thermal stability of the enzyme, possibly because of local conformational flexibility on the dimer interface. The T75C mutant, like the wild‐type enzyme, was inactivated by NADPH, proving that this inactivation cannot be due to simple dissociation of the dimer.