Mutation of conserved residues in Escherichia coli thioredoxin: effects on stability and function.
Mutations were made in three highly conserved residues in Escherichia coli thioredoxin. An internal charged residue, Asp-26, was changed to an alanine. The mutant protein was more stable than the wild type. It can function as a substrate for thioredoxin reductase with a 10-fold increase in the Km over the wild type. Although the redox potential was not substantially changed from that of the wild type, thioredoxin D26A was a poor reducing agent for ribonucleotide reductase. Asp-26 apparently serves to maintain an optimal charge distribution in the active site region for interaction with other proteins. Mutation of a surface Pro-34 in the active site disulfide ring to a serine had little effect on protein stability. A slight decrease in the redox potential (9 mV) made thioredoxin P34S a better reducing agent for ribonucleotide reductase. In contrast, mutation of the internal cis Pro-76 to an alanine destabilized the protein. The data indicate a change had also occurred in the charge distribution in the active site region. Thioredoxin P76A had a higher redox potential than the wild type protein and was not an effective reducing agent for ribonucleotide reductase. It was concluded that this residue is essential for maintaining the conformation of the active site and the redox potential of thioredoxin.
Gleason
F K
FK
Department of Plant Biology, University of Minnesota, St. Paul 55108.
D000595:Amino Acid Sequence; D017124:Conserved Sequence / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology*; D005453:Fluorescence; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011489:Protein Denaturation; D012262:Ribonucleoside Diphosphate Reductase / Q000378:metabolism; D013879:Thioredoxins / Q000235:genetics* / Q000378:metabolism*
D013879:Thioredoxins; D012262:Ribonucleoside Diphosphate Reductase