Disulfide bond intermediates

The ability to trap disulfide-bonded intermediates in the folding pathway of bovine pancreatic trypsin inhibitor (BPTl) enabled Creighton to... 

Bovine pancreatic trypsin inhibitor (BPTI), a small protein with 56 amino acid residues (Fig. 7), is the first one for which a detailed map of the refolding pathways was deciphered. The native state of BPTI contains three disulfide (S-S) bonds formed between six Cys residues. Native state is specified by [30-51 5-55 14—38] bonds. This notation indicates that Cys forms an S-S bond with Cys, and so on. Reduction of the S-S bonds unfolds BPTI. By using S-S bond formation as a progress variable, Creighton [79-83] devised ingenious methods to trap the disulfide-bonded intermediates along the folding pathway. 

To clarify the relevance of non-native intermediates in the folding of proteins dictated by the formation of disulfide bonds Camacho and Thirumalai [45] used lattice models. While these models are merely caricatures of proteins, they contain the specific effects that can be studied in microscopic detail. We used a two-dimensional lattice sequence consisting of hydrophobic (H), polar (P), and Cys (C) residues. If two C beads are near neighbors on the lattice, they can form a S-S bond with an associated energy gain of —with > 0. Thus, topological specificity is required for native S-S bond formation in this model. We have studied the folding kinetics of this model, which is perhaps the simplest model that can probe the characteristics of native and non-native disulfide bonded intermediates. 

Fig. 8.8. Apparent energetic diagrams of the intramolecular transitions in the foldingunfolding of BPTL Species I and II refer to the single and two-disulfide bond intermediates (see Fig. 8.7). The relative free energies of the reduced and native states depend on the stabilities...

Fig. 9.14. Competition between I]-BPTI and reduced intermediates and refolded forms of BPTI for anti-N-antibodies (A) (0)i native ( ), refolded (A), reduced-carboxymethylated BPTI (B) (A) single-disulfide bond intermediate (5-30) and (A)> (30-51) (C) (O) two-disulfide bond intermediate (30-51,14-38) and (A), the mixture of (30-51,5-14) and (30-51,5-38) and (D) (A) nativelike two-disulfide bond species (30-51, 5-55) and (A) N cm and (O) N gJS (from Creighton et al., 1978, courtesy of Creighton).

Human proinsulin has been synthesized in homogeneous solution from 11 protected fragments using azide coupling.1151 The difficulties with insoluble intermediates were sufficiently overcome to allow the 86-residue peptide to be synthesized. The product was de-protected, converted into the 5-sulfonate, and then reduced and reoxidized to form the three disulfide bonds. The product was extensively purified and analyzed, and shown to be pure proinsulin. This product could then be converted into insulin by the use of endopeptidases I and II from pancreatic (3-cell granules, together with carboxypeptidase H, which removed the four basic residues 31, 32, 64, and 65, and split out C-peptide.1 6 ... 

The advantages of this approach are the opportunity to purify the intermediate S-protected derivatives, the introduction of the disulfide bonds in the early stages of the synthesis/62 and also a one-pot deprotection/resin cleavage/oxidation method/63-65 The main reagents used for this purpose are summarized in Table 2. 

A reasonable mechanism for the iodine oxidation of 5-Trt cysteine peptides is given in Scheme 6. 45 Reaction of iodine with the divalent sulfur atom leads to the iodosulfonium ion 5 which is then transformed to the sulfenyl iodide 6 and the trityl cation. Sulfenyl iodides are also postulated as intermediates in the iodine oxidation of thiols to disulfides. The disulfide bond is then formed by disproportionation of two sulfenyl iodides or by reaction between the electrophilic sulfur atom of R -S-I and the nucleophilic S-atom of a second R -S-Trt molecule. The proposed mechanism suggests that any sulfur substitution (i.e., thiol protecting group) capable of forming a stabilized species on cleavage, such as the trityl cation, can be oxidatively cleaved by iodine. 

This method is based on the activation of one cysteine peptide as a sulfenohydrazide derivative by the reaction of the free thiol group of the cysteine peptide with azodicarboxylic acid as a dialkyl ester or dimorpholide depending on the solubility requirements.156 Following the isolation and characterization of the S-activated intermediate, reaction with a second free thiol cysteine component via thiolysis leads to the desired intermolecular disulfide bond, as shown in Scheme 4. 

In the first step of the conversion catalyzed by pyruvate decarboxylase, a carbon atom from thiamine pyrophosphate adds to the carbonyl carbon of pyruvate. Decarboxylation produces the key reactive intermediate, hydroxyethyl thiamine pyrophosphate (HETPP). As shown in figure 13.5, the ionized ylid form of HETPP is resonance-stabilized by the existence of a form without charge separation. The next enzyme, dihydrolipoyltransacetylase, catalyzes the transfer of the two-carbon moiety to lipoic acid. A nucleophilic attack by HETPP on the sulfur atom attached to carbon 8 of oxidized lipoic acid displaces the electrons of the disulfide bond to the sulfur atom attached to carbon 6. The sulfur then picks up a proton from the environment as shown in figure 13.5. This simple displacement reaction is also an oxidation-reduction reaction, in which the attacking carbon atom is oxidized from the aldehyde level in HETPP to the carboxyl level in the lipoic acid derivative. The oxidized (disulfide) form of lipoic acid is converted to the reduced (mer-capto) form. The fact that the two-carbon moiety has become an acyl group is shown more clearly after dissocia-... 

Sodium tetrathionate (Na2S406) is a redox compound that under the right conditions can facilitate the formation of disulfide bonds from free sulfhydryls. The tetrathionate anion reacts with a sulfhydryl to create a somewhat stable active intermediate, a sulfenylthiosulfate (Fig. 102). Upon attack of the nucleophilic thiolate anion on this activated species, the thiosulfate (S203 =) leaving group is removed and a disulfide linkage forms (Pihl and Lange, 1962). The reduction of tetrathionate to thiosulfate in vivo was a subject of early study (Theis and Freeland, 1940 Chen et al., 1934). 

Wang, H., Parry, D. A. D., Jones, L. N., Idler, W. W., Marekov, L. N., and Steinert, P. M. (2000). In vitro assembly and structure of trichocyte keratin intermediate filaments A novel role for stabilization by disulfide bonding./. Cell Biol. 151, 1459-1468. 

Sulfur free-radical chemistry is largely governed by the ability of sulfur to form three-electron bonded intermediates. A case in point is the complexation of a thiyl radical with a thiolate ion (for an analogy with the halide and other pseudohalide systems, see Chap. 5.2). These disulfide radical anions are characterized by strong absorptions in the UV-Vis (Adams et al. 1967). Complexation can occur both intermolecularly as well as intramolecularly. For GSH, for example, the stability constant of the disulfide radical anion is 2900 dm3 mol1 (Mezyk 1996a). The protonated disulfide radical anion is not stable, but such intermediates are known in the cases of the intramolecular complexes [reactions (39) and (40) Akhlaq and von Sonntag 1987]. 

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