Trypsinogen and chymotrypsinogen: the mysterious hyper‐reactivity of selected cysteines is still present after their divergent evolution. (30th May 2021)

• Record Type: Journal Article
• Authors: Cattani, Giada; Bocedi, Alessio; Gambardella, Giorgia; Iavarone, Federica; Boroumand, Mozhgan; Castagnola, Massimo; Ricci, Giorgio
• Is Part Of: FEBS journal. Volume 288:Number 20(2021)
• Page Start: 6003
• Page End: 6018
• Publication Date: 2021-05-30
• Subjects: chymotrypsinogen -- cysteine reactivity -- molten globule -- oxidative folding -- trypsinogen; Biochemistry -- Periodicals; Molecular biology -- Periodicals; Pathology, Molecular -- Periodicals; 572
• DOI: 10.1111/febs.15886
• Languages: English
• ISSNs: 1742-464X

Abstract

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 ( K D = 0.4 mm ) and accelerating the reaction of its cysteines by thousands of times. Abstract : The hyper‐reactivity of cysteines is still present after divergent evolution of trypsinogen and chymotrypsinogen. These proteins show a similar hyper‐reactivity toward oxidized glutathione (GSSG) and other thiol reagents. The enhanced reactivity of Cys168 and Cys220 in the reduced trypsinogen for the GSSG is about 800 times higher when compared to an unperturbed protein cysteine. Moreover, Cys1 in the reduced chymotrypsinogen shows about 1400 times enhanced reactivity.