Adjacent cysteine residues as a redox switch.
Oxidation of adjacent cysteine residues into a cystine forms a strained eight-membered ring. This motif was tested as the basis for an enzyme with an artificial redox switch. Adjacent cysteine residues were introduced into two different structural contexts in ribonuclease A (RNase A) by site-directed mutagenesis to produce the A5C/A6C and S15C/S16C variants. Ala5 and Ala6 are located in an alpha-helix, whereas Ser15 and Ser16 are located in a surface loop. Only A5C/A6C RNase A had the desired property. The catalytic activity of this variant decreases by 70% upon oxidation. The new disulfide bond also decreases the conformational stability of the A5C/A6C variant. Reduction with dithiothreitol restores full enzymatic activity. Thus, the insertion of adjacent cysteine residues in a proper context can be used to modulate enzymatic activity.
Park
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Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA.
Raines
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D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D002384:Catalysis; D003545:Cysteine / Q000737:chemistry*; D004220:Disulfides; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011487:Protein Conformation; D012259:Ribonuclease, Pancreatic / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000737:chemistry
D004220:Disulfides; D012694:Serine; D012259:Ribonuclease, Pancreatic; D003545:Cysteine; D000409:Alanine; D010100:Oxygen