BACTERIAL PROTEIN DISULFIDE-ISOMERASE - EFFICIENT CATALYSIS OF OXIDATIVE PROTEIN-FOLDING AT ACIDIC PH

Periplasmic protein disulfide isomerase (DsbA) is essential for disulf ide formation in Escherichia coli. The tryptophan fluorescence of DsbA measures the redox state of the enzyme during catalysis of the oxidat ive folding of hirudin, a thrombin inhibitor containing three disulfid e bonds and lacking tryptophan. With stoichiometric amounts of DsbA, r educed hirudin is rapidly oxidized in a process initially leading to r andom disulfides. Disulfide reshuffling involving reduced DsbA yields completely native inhibitor within 1 h, even at pH 4. Catalytic amount s of DsbA become essential for hirudin folding in the presence of redo x buffers at acidic pH. The second-order rate constants of disulfide e xchange reactions involving DsbA are several orders of magnitude above the known values for alkyl dithiols and disulfide oxidoreductases. Ds bA preferably reacts with reduced, unfolded polypeptides. The reductio n of DsbA by hirudin is faster by 1 order of magnitude than its reduct ion by the strong reductant dithiothreitol. Together, unusually fast d isulfide interchange reactions and a preference for folding polypeptid es appear to be responsible for the catalytic efficiency of DsbA and f or disulfide formation in vivo at acidic pH.