Directed mutagenesis of the redox-active disulphide bridge in glutathione reductase from Escherichia coli.
Directed mutagenesis of the gor gene from Escherichia coli encoding the flavoprotein glutathione reductase was used to convert the two cysteine residues that comprise its redox-active disulphide bridge to alanine (C42A) and serine (C47S) residues. A double mutant (C42AH439A) was also created in which His-439, the proton donor/acceptor in the glutathione-binding site, was additionally converted into an alanine residue. The C42A and C47S mutants were both unable to catalyse the reduction of glutathione by NADPH. The C42A mutant retained the transhydrogenase activity of the wild-type enzyme, whereas the C47S mutant was also inhibited in this reaction. These results support the view that in the catalytic mechanism of E. coli glutathione reductase, the thiolate form of Cys-42 acts as a nucleophile to initiate disulphide exchange with enzyme-bound glutathione and that the thiolate form of Cys-47 generates an essential charge-transfer complex with enzyme-bound FAD. Titration of the C42A and C42AH439A mutants indicated that the imidazole side-chain of His-439 lowered the pKa of the charge-transfer thiol (Cys-47) from 7.7 to 5.7, enhancing its ability to act as an anion at neutral pH. Several important differences between these mutants of E. coli glutathione reductase and similar mutants (or chemically modified forms) of other members of the flavoprotein disulphide oxidoreductase family were noted, but these could be explained in terms of the different redox chemistries of the enzymes concerned.
Deonarain
M P
MP
Department of Biochemistry, University of Cambridge, U.K.
Scrutton
N S
NS
Berry
A
A
Perham
R N
RN
D000409:Alanine; D001483:Base Sequence; D001665:Binding Sites; D003545:Cysteine; D004220:Disulfides / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005980:Glutathione Reductase / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015345:Oligonucleotide Probes; D010084:Oxidation-Reduction; D010957:Plasmids; D013053:Spectrophotometry
D004220:Disulfides; D015345:Oligonucleotide Probes; D005980:Glutathione Reductase; D003545:Cysteine; D000409:Alanine