Revised Manuscript Received May 4, 1989 abstract: Protein disulfide-isomerase, a protein localizedto the lumen of the endoplasmic reticulum of eukaryotic cells, catalyzes theposttranslational formation and rearrangment of protein disulfide bonds. As isolated from bovine liver, the enzyme contains 0.8 free sulfhydryl groupper mole of protein monomer and 3.1 disulfide bonds. Single-turnover experiments in which the disulfide bonds of the native enzyme are reduced by glutathione reveal three distinct reduction steps corresponding to the sequential reduction of the three disulfide bonds. The fastest disulfide to be reduced undergoes a change in the rate-determining step with increasing GSH concentration from a step which is second-order with respect to GSH concentration to a step which is first-order in GSH concentration. The disulfide which is reduced at an intermediate rate displays kinetics that are first-order in GSH concentration, and the slowest disulfide to be reduced exhibits kinetics which are second-order inGSH concentration. Theenzyme catalyzes the steady-state reduction of a disulfide-containing hexapeptide (CYIQNC) by GSH. Initial velocity kinetic experiments are consistent with a sequential addition of the substrates to the enzyme. Saturation behavior is not observed at high levels of bothsubstrates {Km for GSH» 14 mM, Km for CYIQNC» 1 mM). Only one of the three disulfides appears to be kinetically competent in the steady-state reduction of CYIQNC by GSH. The second-order thiol/disulfide exchange reactions catalyzed by the enzyme are 400-6000-fold faster thanthe corresponding uncatalyzed reactions. iRotein disulfide-isomerase (PDI) 1 is a 56 800-kDa protein which appears to be located primarily in the lumen of the endoplasmic reticulum of eukaryotic cells (Freedman, 1984; Hillson et al., 1984). PDI is thought to catalyze thiol/disulfide exchange reactions involved in the posttranslationalformation of disulfide bonds (Freedman, 1984; Hillson et al., 1984;