New Factors Enhancing the Reactivity of Cysteines in Molten Globule-Like Structures

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.

Trypsinogen and chymotrypsinogen: the mysterious hyper‐reactivity of selected cysteines is still present after their divergent evolution

“…As recently demonstrated for rChTg and other proteins displaying hyper‐reactivity toward various thiol reagents [17], the effect of a lowered p K a of the sulfhydryl group of a protein cysteine cannot produce increased reactivity higher than three to four times at physiological pH values [17]. Thus, the observed hyper‐reactivity toward these hydrophobic reagents has been reasonably attributed to a prominent hydrophobic interaction (about 98%) (Fig.…”

Oxidative Folding of Proteins: The “Smoking Gun” of Glutathione

“…These data suggest a strong hyper-reactivity of Cys18 and other cysteines whose cause remains a fascinating enigma to be solved in the future. This property resembles the recently discovered hyper-reactivity toward GSSG of specific cysteines in the molten globular structures of albumin, lysozyme, ribonuclease trypsinogen, and chymotrypsinogen [ 8 , 9 , 10 , 11 , 12 ] but its origin is likely different. In fact, in the exit tunnel no globular structure of the protein can exist, thus no active-site-like cavity may be able to bind GSSG as it occurs in the molten globules of the above cited proteins.…”

“… Dependence of the second-order kinetic constants (α k RS -) on p K a for the reaction of several thiols with different p K a with different disulfides at pH 7.4 (modified from Ref. [ 9 ]). The red arrow marks the maximum value of the bell-shaped graph.…”

“…Recently, we found that a few cysteines in the fully reduced albumin, adopting a molten globule-like conformation, showed unusual hyper-reactivity toward GSSG and various thiol reagents [ 8 ]. In particular, a single cysteine, identified as Cys75, displayed a second-order kinetic constant > 250 M −1 s −1 which corresponds to more than one thousand times higher reactivity toward GSSG than the one of an unperturbed protein cysteine ( k = 0.2 M −1 s −1 ) ( Figure 1 ) [ 9 ]. At first, we considered this surprising reactivity as a specific feature of a single protein.…”

“…A possible role in this phenomenon of a lowered p K a of the sulfhydryl group was also considered but a recent investigation [ 9 ] likely demonstrated that a low p K a cannot produce more than three times increased reactivity toward GSSG ( Figure 2 ).…”