title: Involvement of two sulfur atoms of protein disulfide isomerase and one sulfur atom of the DsbA/PpfA protein in the oxidation of mutant human lysozyme.
abstract: Protein disulfide isomerase (PDI) and the DsbA/PpfA protein catalyze the oxidation of mutant human lysozyme, L79CC81A, which has two native disulfide bonds, Cys6-Cys128 and Cys30-Cys116, a non-native Cys79-Cys95, and 2 free cysteine residues at positions 65 and 77. Oxidation of L79CC81A (R-form) yielded two isomers, L79CC81A-a (A-form) with tandem-linked Cys65-Cys77 and Cys79-Cys95, and L79CC81A-b (B-form) with cross-linked Cys65-Cys79 and Cys77-Cys95 (Kanaya, E., Ishihara, K., Tsunasawa, S., Nokihara, K., and Kikuchi, M. (1993) Biochem. J. 292, 469-476). PDI mainly enhanced the formation of the A- form in the absence of oxidized glutathione (GSSG); however, as the concentration of GSSG increased, it markedly accelerated the formation of the B-form. In contrast, the DspA/PpfA protein mainly enhanced the formation of the A-form, regardless of the presence or absence of GSSG. These results and the presumed spatial locations of Cys65, Cys77, and Cys79-Cys95 in the R-form suggest that 1 of the half-cystine residues in the active site of PDI and the DsbA/PpfA protein can react with 1 of the 2 free Cys residues of the R-form. The dependence on GSSG of the B-form formation with PDI can be explained by the formation of two transient intermolecular disulfide bonds between PDI and the R-form and the attack of GSSG by the resultant thiolate anion of Cys79 or Cys95. The independence of the reaction with the DsbA/PpfA protein from GSSG can be explained by the formation of one transient intermolecular disulfide bond. The possible formation of the two transient intermolecular disulfide bonds involving two sulfur atoms of PDI and 2 cysteine or half-cystine residues of the substrate could explain the high isomerase activity of PDI.
authors.affiliation: Protein Engineering Research Institute, Osaka, Japan.
authors.forename: E
authors.initials: E
authors.lastname: Kanaya
authors.forename: H
authors.initials: H
authors.lastname: Anaguchi
authors.forename: M
authors.initials: M
authors.lastname: Kikuchi
mesh_terms: D001665:Binding Sites; D004220:Disulfides / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D007535:Isomerases / Q000737:chemistry*; D007536:Isomerism; D009113:Muramidase / Q000378:metabolism*; D010084:Oxidation-Reduction; D019704:Protein Disulfide-Isomerases; D011994:Recombinant Proteins
chemical_list: D004220:Disulfides; D011994:Recombinant Proteins; D009113:Muramidase; D007535:Isomerases; D019704:Protein Disulfide-Isomerases; D005978:Glutathione