The C-terminal active site cysteine of Escherichia coli glutaredoxin 1 determines the glutathione specificity of the second step of peptide deglutathionylation.
Glutaredoxins are oxidoreductases specialized in reducing glutathione-protein mixed disulfides. In the first step of deglutathionylation, glutaredoxins form a mixed disulfide with glutathione, releasing reduced peptide. The specificity of this reaction is based on the unusual amide linkage formed between the gamma-carboxylate of the N-terminal glutamic acid and the alpha-amino group of the cysteine present in glutathione. In the second step of deglutathionylation, glutathione reduces the glutaredoxin-glutathione mixed disulfide. Here we show that the specificity of this second reaction for Escherichia coli Grx1, but not for human or yeast Grx1, also is based on the unusual gamma-linkage present in glutathione. Mutating Tyr13, Thr58, and/or Asp74 to alanine in E. coli Grx1 results in the glutaredoxin-peptide mixed disulfide being thermodynamically favored over the glutaredoxin-glutathione mixed disulfide in the first step of the reaction. An increased propensity to form glutaredoxin-protein mixed disulfides was observed in vivo for these same mutants. Furthermore, we demonstrate that all mutations studied in Cys14, the C-terminal active site cysteine, abolish the specificity of E. coli Grx1 for glutathione over the corresponding tripeptide Glu-Cys-Gly, which has a normal peptide bond linking Glu-Cys instead of the gamma-linkage present in glutathione, in the second step of deglutathionylation.
Saaranen
Mirva J
MJ
Department of Biochemistry, University of Oulu, Oulu, Finland.
Salo
Kirsi E H
KE
Latva-Ranta
Maria K
MK
Kinnula
Vuokko L
VL
Ruddock
Lloyd W
LW
D055162:Biocatalysis; D015153:Blotting, Western; D020134:Catalytic Domain; D017124:Conserved Sequence; D003545:Cysteine / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D054477:Glutaredoxins / Q000737:chemistry / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism*; D006801:Humans; D013058:Mass Spectrometry; D013379:Substrate Specificity
D054477:Glutaredoxins; D005978:Glutathione; D003545:Cysteine