INVOLVEMENT OF 2 SULFUR-ATOMS OF PROTEIN DISULFIDE-ISOMERASE AND ONE SULFUR ATOM OF THE DSBA PPFA PROTEIN IN THE OXIDATION OF MUTANT HUMAN LYSOZYME/

Protein disulfide isomerase (PDI) and the DsbA/PpfA protein catalyze t he oxidation of mutant human lysozyme, L79CC81A, which has two native disulfide bonds, Cys(6)-Cys(128) and Cys(30)-Cys(116), a non-native Cy s(79)-Cys(95), and 2 free cysteine residues at positions 65 and 77. Ox idation of L79CC81A (R-form) yielded two isomers, L79CC81A-a (A-form) with tandem-linked Cys(65)-Cys(77) and Cys(79)-Cys(95), and L79CC81A-b (B-form) with crosslinked Cys(65)-Cys(79) and Cys(77)-Cys(95) (Kanaya , E., Ishihara, It., Tsunasawa, S., Nokihara, K., and Kikuchi, M. (199 3) Biochem. J. 292, 469-476). PDI mainly enhanced the formation of the A- form in the absence of oxidized glutathione (GSSG); however, as th e concentration of GSSG increased, it markedly accelerated the formati on of the B-form. In contrast, the DspA/PpfA protein mainly enhanced t he formation of the A-form, regardless of the presence or absence of G SSG. These results and the presumed spatial locations of Cys(65), Cys( 77), and Cys(79)-Cys(95) in the R-form suggest that 1 of the half-cyst ine residues in the active site of PDI and the DsbA/PpfA protein can r eact with 1 of the 2 free Cys residues of the R-form. The dependence o n GSSG of the B-form formation with PDI can be explained by the format ion of two transient in termolecular disulfide bonds between PDI and t he R-form and the attack of GSSG by the resultant thiolate anion of Cy s(79) or Cys(95). The independence of the reac tion 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 trans ient 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.