A mutant thioredoxin from Escherichia coli tsnC 7007 that is nonfunctional as subunit of phage T7 DNA polymerase.
Thioredoxin was purified to homogeneity from the Escherichia coli mutant tsnC 7007 that is defective in phage T7 DNA replication and previously shown to contain a missense thioredoxin. Tryptic peptide maps of reduced and carboxymethylated 7007 thioredoxin combined with amino acid sequence analysis revealed one amino acid substitution; Gly-92 in thioredoxin is exchanged to an aspartic acid residue in the 7007 protein. The missense thioredoxin gave no activity with the gene 5 protein of phage T7 in the complementation to active T7 DNA polymerase. It competitively inhibited the complementation of wild type thioredoxin and gene 5 protein and formed a complex with the gene 5 protein that was retained by antithioredoxin Sepharose. The 7007 thioredoxin has reduced catalytic activity with thioredoxin reductase, ribonucleotide reductase, or as a protein disulfide reductase. The apparent Km value of 7007 thioredoxin as a substrate for thioredoxin reductase was increased 3-fold relative to normal thioredoxin, and the Vmax value was decreased 7-fold. The position of GLy-92 in the known three-dimensional structure of thioredoxin-S2 is correlated with the changed functional properties of the substituted mutant protein.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001426:Bacterial Proteins / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D007122:Immunoelectrophoresis; D007700:Kinetics; D046911:Macromolecular Substances; D008958:Models, Molecular; D009154:Mutation; D010446:Peptide Fragments / Q000032:analysis; D011487:Protein Conformation; D013604:T-Phages / Q000201:enzymology*; D013879:Thioredoxins / Q000302:isolation & purification / Q000378:metabolism*; D014357:Trypsin
D000596:Amino Acids; D001426:Bacterial Proteins; D046911:Macromolecular Substances; D010446:Peptide Fragments; D013879:Thioredoxins; D004259:DNA-Directed DNA Polymerase; D014357:Trypsin