IONIZATION-REACTIVITY RELATIONSHIPS FOR CYSTEINE THIOLS IN POLYPEPTIDES

Thiol-disulfide exchange reactions are required for many aspects of ce llular metabolism including the folding of disulfide-bonded proteins, electron transfer, and numerous regulatory mechanisms. To identify fac tors influencing the rates of these reactions in polypeptides, the rea ctivities of Cys thiols in 16 model peptides were measured. For each o f the peptides, which contained single Cys residues with thiol pK(a)s ranging from 7.4 to 9.1, the rates of exchange with four disulfide-bon ded compounds were measured. In reactions with two of the disulfide re agents, cystine and 2-hydroxyethyl disulfide, the peptide thiols displ ayed Bronsted correlations between reaction rate and pK(a) similar to those observed previously with model compounds (beta(nuc) = 0.5 and 0. 3, respectively). For two reagents with net charges, oxidized glutathi one and cystamine, however, the apparent Bronsted coefficients were 0 and 0.8, respectively. These observations are in striking contrast wit h those obtained with model compounds, for which the Bronsted coeffici ents for the nucleophilic thiolates are largely independent of the dis ulfide-containing compound. The differences in the apparent Bronsted c oefficients can be largely accounted for by electrostatic interactions between charged groups on the peptides and disulfide reagents and dem onstrate that such interactions can play a dominant role in determinin g the rates of thiol-disulfide exchange in biological molecules. The r esults presented here provide an improved basis for predicting the rat es of these reactions and suggest ways in which differences in the rat es of competing reactions can be either minimized, to simplify the ana lysis of disulfide-coupled folding reactions, or enhanced, to favor fo rmation of particular disulfides.