Abstract
Protein cysteines often play crucial functional and structural roles, so they are emerging targets to design covalent thiol ligands that are able to modulate enzyme or protein functions. Some of these residues, especially those involved in enzyme mechanisms—including nucleophilic and reductive catalysis and thiol-disulfide exchange—display unusual hyper-reactivity; such a property is expected to result from a low pKa and from a great accessibility to a given reagent. New findings and previous evidence clearly indicate that pKa perturbations can only produce two–four-times increased reactivity at physiological pH values, far from the hundred and even thousand-times kinetic enhancements observed for some protein cysteines. The data from the molten globule-like structures of ribonuclease, lysozyme, bovine serum albumin and chymotrypsinogen identified new speeding agents, i.e., hydrophobic/electrostatic interactions and productive complex formations involving the protein and thiol reagent, which were able to confer exceptional reactivity to structural cysteines which were only intended to form disulfides. This study, for the first time, evaluates quantitatively the different contributions of pKa and other factors to the overall reactivity. These findings may help to clarify the mechanisms that allow a rapid disulfide formation during the oxidative folding of many proteins.
Highlights
Protein cysteines are involved in many critical structural and functional roles that can be performed thanks to the peculiar properties of their sulfhydryl group, which, in its deprotonated form, becomes an active nucleophile
A different and opposite behavior was observed for the reduced ribonuclease in its reactions with CDNB and NBD-Cl at different ionic strengths (Figure 4C)
The conversion of the native oxidized form of albumin, chymotrypsinogen and lysozyme into a reduced molten globule-like structure causes a loss of the alpha helix and an accompanying increase in the beta sheet content. This evidence only suggests some changes of the secondary structures, but nothing about an increased exposition of hydrophobic residues which are able to promote a productive binding of CDNB and NBD-Cl
Summary
Protein cysteines are involved in many critical structural and functional roles that can be performed thanks to the peculiar properties of their sulfhydryl group, which, in its deprotonated form, becomes an active nucleophile. The values of the Brønsted coefficient (βnuc) and C (a constant applicable to a specific reaction involving various thiols and alkylating reagents) were 0.46 ± 0.07 (βnuc) and −3.99 ± 0.56 (C) for CDNB and 0.53 ± 0.08 (βnuc) and −2.13 ± 0.67 (C) for NBD-Cl at pH = 7.4 These values were inserted into the Equation (6), obtaining a bell-shaped behavior with a maximum at pKa ≈ 7.4 (Figure 1A,B). A similar variation of the nucleophilicity of the sulfhydryl group was observed during the reaction of a few disulfides with free thiols or cysteine containing peptides showing different pKa. Even in that case, a linear decrease was found by plotting the logarithm of the second order kinetic constant of the thiolate onto the corresponding pKa of the thiols [9,10,11,12]. Our experimental data, together with preceding results, indicate that protein cysteines showing pKa ≤ 5.0 are almost unreactive toward various thiol reagents at physiological pH values
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