Ionization−Reactivity Relationships for Cysteine Thiols in Polypeptides

Abstract

Thiol-disulfide exchange reactions are required for many aspects of cellular metabolism including the folding of disulfide-bonded proteins, electron transfer, and numerous regulatory mechanisms. To identify factors influencing the rates of these reactions in polypeptides, the reactivities of Cys thiols in 16 model peptides were measured. For each of the peptides, which contained single Cys residues with thiol pKas ranging from 7.4 to 9.1, the rates of exchange with four disulfide-bonded compounds were measured. In reactions with two of the disulfide reagents, cystine and 2-hydroxyethyl disulfide, the peptide thiols displayed Bronsted correlations between reaction rate and pKa similar to those observed previously with model compounds (betanuc = 0.5 and 0.3, respectively). For two reagents with net charges, oxidized glutathione and cystamine, however, the apparent Bronsted coefficients were 0 and 0.8, respectively. These observations are in striking contrast with those obtained with model compounds, for which the Bronsted coefficients for the nucleophilic thiolates are largely independent of the disulfide-containing compound. The differences in the apparent Bronsted coefficients can be largely accounted for by electrostatic interactions between charged groups on the peptides and disulfide reagents and demonstrate that such interactions can play a dominant role in determining the rates of thiol-disulfide exchange in biological molecules. The results presented here provide an improved basis for predicting the rates of these reactions and suggest ways in which differences in the rates of competing reactions can be either minimized, to simplify the analysis of disulfide-coupled folding reactions, or enhanced, to favor formation of particular disulfides.

Catalytic Properties of the Archaeal S-Adenosylmethionine Decarboxylase from Methanococcus jannaschii

“…The pK near 8.2 is in the normal range of a cysteine sulfhydryl in peptides (33). By analogy to previous studies of eucaryotic AdoMetDC, this pK likely reflects the ionization of cysteine in the conserved Thr-Cys-Gly sequence located 19 residues C-terminal to the pyruvoyl group in the M. jannaschii protein and in similar locations in all other characterized AdoMet decarboxylases (17,34).…”

“…It has been proposed that this cysteine acts to protonate the ␣-carbon of the product Schiff base, a role consistent with its location in the crystal structure of the human enzyme (15). A pK near 8 is typical for a cysteine sulfhydryl in peptides (33). The pK values for the groups that must be deprotonated in the k cat profile are 6.3 (human), 6.7 (E. coli), and 5.3 (M. jannaschii); in the k cat /K m profile, the corresponding pK values are 6.9 (human), 6.7 (E. coli), 6.5 (M. jannaschii), and 7.8 (T. cruzi, 2 protons).…”

Combination of resveratrol and fluoxetine in an acute model of depression in mice: Prevention of oxidative DNA fragmentation and monoamines degradation

“…The homogenate was divided into 2 portions for the determination of MDA (Ruiz-Larrea et al, 1994) and GSH (Bulaj et al, 1998, Ellman, 1959.…”

Delivery of antigen using a novel mannosylated dendrimer potentiates immunogenicity in vitro and in vivo

“…The solution was then dialyzed overnight into PBS/2 mM EDTA at 4 C with 6000-8000 MWCO tubing to remove excess TCEP from the solution. The sulfhydryl groups were quantitated as previously described [42,43]. OVA was modified by the addition of 18 lL 20 mg/mL SPDP (Pierce) in DMSO to 500 lL 10 mg/mL OVA in PBS, and mixed for 16 h at room temperature to generate OVA-SPDP.…”