The N-terminal sequence (residues 1-65) is essential for dimerization, activities, and peptide binding of Escherichia coli DsbC.
Limited proteolysis of DsbC with trypsin resulted in a compact and stable C-terminal fragment (residues 66-216), fDsbC, which retains the active site sequence, -Cys(98)-Gly-Tyr-Cys(101)-, and shows only minor differences in conformation compared with that of the intact molecule. The pK(a) of active site thiol and the K(SS) with glutathione are very close to that of DsbC, respectively; however, fDsbC is inactive as an isomerase in catalyzing the formation of correct disulfide bonds in scrambled RNase A and denatured and reduced bovine pancreatic trypsin inhibitor and shows only 13% thiol-protein oxidoreductase activity (TPOR) of DsbC. In contrast to the dimeric DsbC, fDsbC exists as a monomer and has no chaperone activity in assisting the reactivation of denatured d-glyceraldehyde-3-phosphate dehydrogenase. The heterodimer of DsbC with the inactive DsbC carboxymethylated at both active site thiols shows about 50% TPOR activity of DsbC but no isomerase activity, indicating that the DsbC subunit in the heterodimer displays full TPOR activity but little, if any, isomerase activity. It is concluded that the N-terminal sequence (residues 1-65) is essential for dimer formation and chaperone activity of DsbC. The active sites in both subunits of the dimeric DsbC appear to be essential for its isomerase activity.
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National Laboratory of Biomacromolecules, Institute of Biophysics, Academia Sinica, Beijing 100101, China.
Wang
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D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D019281:Dimerization; D004926:Escherichia coli / Q000378:metabolism*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism*; D019704:Protein Disulfide-Isomerases / Q000737:chemistry / Q000378:metabolism*; D012259:Ribonuclease, Pancreatic / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D010446:Peptide Fragments; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D012259:Ribonuclease, Pancreatic; D014357:Trypsin; D019704:Protein Disulfide-Isomerases