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

Abstract

An enigmatic and never described hyper‐reactivity of most of the cysteines resident in the reduced, molten globule‐like intermediate of a few proteins has been recently discovered. In particular, all ten cysteines of chymotrypsinogen showed hundred times increased reactivity against hydrophobic reagents. A single cysteine (Cys1) was also found thousand times more reactive toward GSSG, making speculate that a single glutathionylation could represent the primordial event of its oxidative folding. In the present study, we compare these kinetic properties with those present in trypsinogen taken in its reduced, molten globule‐like intermediate and identify the origin of these unusual properties. Despite the divergent evolution of these two proteins, the different amount of disulfides and the very different 3D localization of three disulfides, their hyper‐reactivity toward hydrophobic thiol reagents and disulfides is very similar. Mass spectrometry identifies two cysteines in trypsinogen, Cys148 and Cys197, 800 times more reactive toward GSSG than an unperturbed protein cysteine. These results point toward a stringent and accurate preservation of these peculiar kinetic properties during a divergent evolution suggesting some important role, which at the present can only be hypothesized. Similar extraordinary hyper‐reactivity has been found also in albumin, ribonuclease, and lysozyme confirming that it cannot be considered a kinetic singularity of a single protein. Interestingly, the very flexible and fluctuating structures like those typical of the molten globule status prove capable of enabling sophisticated actions typical of enzymes such as binding to GSSG with relevant specificity and high affinity (KD = 0.4 mm) and accelerating the reaction of its cysteines by thousands of times.

The unusual properties of lactoferrin during its nascent phase

“…At physiological pHs this protein undergoes insoluble aggregates. We observed a similar phenomenon in other reduced proteins like lysozyme, chymotrypsinogen and trypsinogen 6 , 8 , 9 . In the case of lysozyme, the rapid glutathionylation of its hyper-reactive cysteine (Cys94) lowered its irreversible aggregation.…”

“…The present study discovers unusual properties of LF in its reduced molten globule-like structure which approaches to the transient architecture of this protein in its nascent phase. As observed in albumin 5 , another protein with many disulfides, as well as in lysozyme 6 , ribonuclease 7 , trypsinogen 8 and chymotrypsinogen 9 all having less disulfides, we found in LF a few structural cysteines, all devoted to form disulfides, displaying extraordinary and specific reactivity toward oxidized glutathione (GSSG). This finding suggests that these residues give mixed disulfide with GSH in the early phase of its oxidative folding.…”

The Anfinsen Dogma: Intriguing Details Sixty-Five Years Later

“…In conclusion, this study describes new details of the in vitro oxidative pathway described many years ago by Anfinsen, but we are aware that the in vivo process can proceed in different ways. The recent discovery of an ultra-rapid glutathionylation of Cys95 [ 31 , 32 ] when reduced RNase is in the state of molten globule and similar phenomenon found in other proteins (i.e., serum albumin, lysozyme, chymotrypsinogen and trypsinogen) [ 33 , 34 , 35 , 36 ] are an intriguing indication in that direction.…”