Revised Manuscript Received October 16, 1995® abstract: The dithiol/disulfide active sites of each of the twoisolated thioredoxin-like domains of protein disulfide isomerase (PDI) expressed in Escherichia coli have been characterized in order to understand their catalytic mechanisms and their functions in PDI. In each of the folded domains, as in other proteins of the thioredoxin family, only one of the cysteine residues of the active site sequence-Cys-Gly-His-Cys-is accessible, and its thiol group is highly reactive and has a low pAfavalue. The kinetics and equilibria have been measured of the reactions between the active site cysteine residues and glutathione, the predominant thiol/disulfide reagent of the endoplasmic reticulum. A disulfide bond can be formed very rapidly between the pair of cysteine residues of each domain, but each disulfide bond is very unstable and reacts rapidly with reduced glutathione. The verylow stabilities of these disulfide bonds, which destabilize the protein structures, account for the efficiency with which PDI and each of the isolated domains can introduce disulfide bonds into proteins. These kinetic and equilibrium data go farin helping to understand the catalytic mechanism of PDI and its individual domains.

Protein disulfide bond formation is an important co-and post-translational event in the biosynthesis of many proteins and is often linked to their folding. In the endoplasmic reticulum of eukaryotic cells, in the presence of millimolar concentrations of both GSH* 1 and GSSG, this process is catalyzed by protein disulfide isomerase [reviewed by Noiva and Lennarz (1992), Freedman (1992, 1995), and Freedman et al.(1994)]. PDI accelerates the rates of disulfide bond formation, breakage, and rearrangementin proteins, steps that can be rate-limiting in disulfide-coupled folding (Givol et al., 1964; Creighton et al., 1980, 1993; Zapun et al., 1992; Darby et al., 1994). The detailed mechanism of how PDI functions is not known, but its primary structure contains two segments that are homologous to thioredoxin, each with a-Cys-Gly-His-Cys-sequence (Edman et al., 1985). It is clear that the function of PDI involves thiol—disulfide exchange reactions at these cysteine residues (Hawkins & Freedman, 1991; Vuori et al., 1992; LaMantia & Lennarz, 1993; Lyles & Gilbert, 1994). In the course of these reactions, it is likely that the active site cysteine residues function in a way similar to that originally proposed for thioredoxin (Kallis & Holmgren, 1980) and are cycled between their thiol and disulfide forms, via a mixed disulfide with the protein substrate or with glutathione (see eqs 1—3 below).