OXIDATION OF KINETICALLY TRAPPED THIOLS BY PROTEIN DISULFIDE-ISOMERASE

The formation of a stabilized structure during oxidative protein foldi ng can severely retard disulfide formation if the structure must be di srupted to gain access to buried cysteines. These kinetic traps can sl ow protein folding and disulfide bond formation to the extent that una ssisted folding is too slow to be kinetically competent in the cell. P rotein disulfide isomerase (PDI) facilitates the oxidation of a kineti cally trapped state of RTEM-1 beta-lactamase in which two cysteines th at form the single disulfide bond in the native protein are buried and approximately 500-fold less reactive than exposed cysteines. Under se cond-order conditions, PDI-dependent oxidation of reduced, folded beta -lactamase is 500-fold faster than GSSG-dependent oxidation. The rate difference observed between PDI and GSSG can be accounted for by the 5 20-fold higher kinetic reactivity of PDI as an oxidant. Noncovalent in teractions between PDI (35 mu M) and beta-lactamase increase the react ivity or unfolding of beta-lactamase in the steady-state by less than 3-fold. At high concentrations of PDI or alkylating agents, the reacti on of beta-lactamase cysteines approaches a constant rate, limited by the spontaneous unfolding of the protein (k(unfold) = 0.024 +/- 0.005 min(-1)). PDI does not substantially increase the rate of beta-lactama se unfolding; however, once beta-lactamase spontaneously unfolds. PDI at concentrations greater than 44 +/- 4 mu M, oxidizes the unfolded su bstrate before it can refold (k(fold) = 1.5 +/- 0.2 min(-1)). PDI also facilitates the glutathione disulfide-dependent oxidation of beta-lac tamase by approximately 1.7-3-fold, even at saturating GSSG concentrat ions; however, oxidation rates never exceed the rate of spontaneous be ta-lactamase unfolding. The high kinetic reactivity of PDI as an oxida nt and the almost millimolar concentrations of PDI found in the endopl asmic reticulum provide one mechanism to avoid kinetic traps by making oxidation kinetically competitive with the formation of kinetic traps . In addition, PDI's high reactivity and its noncovalent interactions with unfolded proteins may minimize the significance of even relativel y stable kinetic traps, as long as unfolding of the kinetic trap does not become rate-limiting. Chaperones, including the hsp70 chaperone of the endoplasmic reticulum, BiP, and the Escherichia coil chaperonin, GroEL, do not appear to increase the rate of beta-lactamase unfolding. This suggests that the unfolding rate may help define those structure s that the endoplasmic reticulum quality control system recognizes as native.