CATALYSIS OF OXIDATIVE PROTEIN-FOLDING BY MUTANTS OF PROTEIN DISULFIDE-ISOMERASE WITH A SINGLE ACTIVE-SITE CYSTEINE

Protein disulfide isomerase (PDI), a very abundant protein in the endo plasmic reticulum, facilitates the formation and rearrangement of disu lfide bends using two nonequivalent redox active-sites, located in two different thioredoxin homology domains [Lyles, M. M., & Gilbert, H. F . (1994) J. Biol. Chem. 269, 30946-30952]. Each dithiol/disulfide acti ve-site contains the thioredoxin consensus sequence CXXC. Four mutants of protein disulfide isomerase were constructed that have only a sing le active-site cysteine. Kinetic analysis of these mutants show that t he first (more N-terminal) cysteine in either active site is essential for catalysis of oxidation and rearrangement during the refolding of reduced bovine pancreatic ribonuclease A (RNase). Mutant active sites with the sequence SGHC show no detectable activity for disulfide forma tion or rearrangement, even at concentrations of 25 mu M. The second ( more C-terminal) cysteine is not essential for catalysis of RNase disu lfide rearrangements, but it is essential for catalysis of RNase oxida tion, even in the presence of a glutathione redox buffer. Mutant activ e sites with the sequence CGHS show 12%-50% of the k(cat) activity of wild-type active sites during the rearrangement phase of RNase refoldi ng but <5% activity during the oxidation phase. In addition, mutants w ith the sequence CGHS accumulate significant levels of a covalent PDI- RNase complex during steady-state turnover while the wild-type enzyme and mutants with the sequence SGHC do not. Since both active-site cyst eines are essential for catalysis of disulfide formation, the dominant mechanism for RNase oxidation may involve direct oxidation by the act ive-site PDI disulfide. Although it is not essential for catalysis of RNase rearrangements, the more C-terminal cysteine does contribute 2-8 -fold to the rearrangement activity. A mechanism for substrate rearran gement is suggested in which the second active-site cysteine provides PDI with a way to ''escape'' from covalent intermediates that do not r earrange in a timely fashion. The second active-site cysteine may norm ally serve the wild-type enzyme as an internal clock that limits the t ime allowed for intramolecular substrate rearrangements.