Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 30 September 2019 doi:10.20944/preprints201909.0341.v1 Peer-reviewed version available at Antioxidants & Redox Signaling 2019; doi:10.1089/ars.2019.7894 Getting the Right Grip? How Understanding Electrophile Selectivity Profiles Could Illuminate Our Understanding of Redox Signaling Marcus J. C. Long†, Lingxi Wang‡, and Yimon Aye‡,* ‡Swiss Federal Institute of Technology Lausanne (EPFL), 1015, Lausanne, Switzerland †47 Pudding Gate, Bishop Burton, Beverley East Riding of Yorkshire, HU17 8QH, UK *Correspondence: Prof. Yimon Aye. CH A2 397, EPFL, 1015, Lausanne, Switzerland. Tel: +41-(0)79 838 94 47. Fax: +41-(0)21 693 66 50. E-mail: [email protected] Abstract: Significance: Electrophile signaling is coming into focus as a bona fide cell signaling mechanism. The electrophilic regulation occurs typically through a sensing event (i.e., labeling of a protein) and a signaling event (the labeling event having an effect of the proteins activity, association, etc.). Recent advances: Herein, we focus on the first step of this process, electrophile sensing. Electrophile sensing is typically a deceptively simple reaction between the thiol of a protein-cysteine, of which there are around 200,000 in the human proteome, and a Michael acceptor, of which there are numerous flavors, including enals and enones. Recent data overall paint a picture that despite being a simple chemical reaction, electrophile sensing is a discerning process, showing labeling preferences that are often not in line with reactivity of the electrophile. Critical issues: With a view to trying to decide what brings about highly electrophile-reactive protein-cysteines, and how reactive these sensors may be, we discuss aspects of the thermodynamics and kinetics of covalent/non-covalent binding. Data made available by several laboratories indicate that it is likely that specific proteins exhibit highly stereo- and chemoselective electrophile sensing, which we take as good evidence for recognition between the electrophile and the protein prior to forming a covalent bond. Future directions: We propose experiments that could help us gain a better and more quantitative understanding of the 1 © 2019 by the author(s). Distributed under a Creative Commons CC BY license. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 30 September 2019 doi:10.20944/preprints201909.0341.v1 Peer-reviewed version available at Antioxidants & Redox Signaling 2019; doi:10.1089/ars.2019.7894 Long, et al. mechanisms through which sensing comes about. We further extoll the importance of performing more detailed experiments on labeling and trying to standardize the way we assess protein-specific electrophile sensing. Key words: Covalent labeling; stereoselectivity; electrophile signaling; affinity; mechanism; kinetic control 2 Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 30 September 2019 doi:10.20944/preprints201909.0341.v1 Peer-reviewed version available at Antioxidants & Redox Signaling 2019; doi:10.1089/ars.2019.7894 Long, et al. Man’s appreciation for molecular shape, configuration, and topology in biology has had a checkered history. These vacillations are perhaps best exemplified by our understanding of enantiomers, although similar arguments hold for other minor structural changes. By the 1800’s, we had started to understand that many naturally-occurring compounds had the ability to change the orientation of plane- polarized light. The examples included several naturally-derived inorganic substances, like quartz crystals (16), as well as bioactive natural products, such as tartrate (15). In the 1840’s, Louis Pasteur performed his famous experiment separating racemic tartaric acid into different crystalline forms, which transpired to contain the resolved enantiomers of the compound (15). Later, it was noted that the enantiomers of borneol had distinctly-different odors, hinting that molecular recognition was linked to smell, and that our bodies can distinguish effectively between enantiomers (6). By the 1930s, the concept of enantiomorphism in biology had permeated the general conscience. A contemporary detective series writer, Dorothy L. Sayers, even used the concept that naturally-derived poisons are homochiral and synthetic versions (like would be found in a synthetic poison) are racemic as the denouement to her novel “The Documents in the Case” (48). In the book, the “poison” was muscarine, a sugar analog that is an agonist of the muscarinic acetylcholine receptor (23). Yet this appreciation for the differential biological properties of enantiomers did little to prepare scientists for the thalidomide crisis in the 1960s that has left medicinal chemists with the critical knowledge that enantiomers can be divergently bioactive. Ironically, this debacle occurred some 10 years after the observations on the potency of different enantiomers by C. Pfeiffer (5). A silent assumption in the above examples of molecules interacting with biological receptors/enzymes/proteins, is that non-covalent binding is the key driving force of the interaction. Indeed, it is the concept of non-covalent binding and the required spatial recognition between enzyme and substrate (or protein and ligand) that has preoccupied biochemists and biologists arguably since the work of Leonor Michaelis and Maud Menten in 1913, and the development of their eponymous equation (or its precursor, the Henri equation) (11). With our current understanding that ligand recognition is a key determinant of biological processes, it is clear why the different enantiomers of different molecules can exhibit divergent 3 Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 30 September 2019 doi:10.20944/preprints201909.0341.v1 Peer-reviewed version available at Antioxidants & Redox Signaling 2019; doi:10.1089/ars.2019.7894 Long, et al. biological properties, such as having different odors. Because high affinity translates to large binding energy, the stabilization of the system incurred upon ligand binding can also be leveraged to induce changes in stabilization, conformation, association, etc., that can be transmuted into meaningful changes in cellular decision-making. The binding requirements can be so stringent, that even very similar molecules that are present together in the cellular milieu can be suitably differentiated by enzyme active sites, and hence this “chemical noise” is efficiently filtered out. The Michaelis-Menten equation also extends to situations where the enzyme forms a covalent bond to the substrate. The covalent intermediate can be harnessed for numerous possible ends, such as: to promote fission, e.g., in serine proteases; or to promote reaction with an electrophile, e.g., in thymidylate synthetase. Nevertheless, it remains generally true that it is the ability of the enzyme to accommodate the substrate(s) in the active site, and orientate reactive positions to promote reactivity that ultimately synergize to allow an overall selective process to occur. For instance, deubiquitinating enzymes mostly hydrolyze essentially “the same” substrate, i.e., an isopeptide bond formed between ubiquitin’s C-terminus and a protein-lysine. Selectivity is engendered by a combination of recognition of ubiquitin chain linkage, and the protein to which the ubiquitin(s) is(are) bound. But we now understand that there are numerous very simple biological signaling molecules that are proposed to regulate numerous important pathways. These molecules, however, appear to be assumed to have little ability to interact non-covalently with their targets. So how can, and indeed, do these molecules interact selectively? In this opinion piece, we compare and contrast the thermodynamics and kinetics of covalent and non-covalent binding, to try to better understand how reactive electrophiles work, and to try to derive some insight into how selectivity and concepts of biochemistry can be used to begin to interrogate electrophile signaling in more detail. Our ultimate hope is that this work will serve as a reminder of the importance of non-covalent interactions in marshaling even overwhelmingly strong covalent bond-forming events, and further to underline the insight that can be brought to the table by examining stereo- and chemoselectivity in biological systems. 4 Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 30 September 2019 doi:10.20944/preprints201909.0341.v1 Peer-reviewed version available at Antioxidants & Redox Signaling 2019; doi:10.1089/ars.2019.7894 Long, et al. Thermodynamics of covalent binding compared to non-covalent bonding Covalent bond formation and non-covalent bonding are critically different. The bond energy of an sp3-hybridized C–H bond is around 420 kJ mol-1. Even a relatively weak sp3-hybridized bond, where orbital overlap is considered poor, such as C–S, has a bond energy of around 290 kJ mol-1. Alternatively the π- bond in C=C has an energy of 266 kJ mol-1. By contrast, “only” 11.5 kJ mol-1 represents the transition- state stabilization energy required for a reaction to proceed with 99% ee at room temperature. This energy difference is less than 5 times the corresponding available thermal energy. Less stabilization is required as the temperature decreases (8 kJ mol-1 at –78 oC). Even what are considered high affinity, non-covalent -1 binders (typically Kd = 0.1–10 nM, equivalent to ΔGbinding = ~60 to 46 kJ mol ) have binding energies significantly lower than that of particularly weak covalent bonds. Furthermore, to achieve even “nanomolar”