KEAP1 C151 active site catalysis drives electrophilic signaling to upregulate cytoprotective enzyme expression.

Cells mount a detoxification, antioxidant, and anti-inflammatory response to electrophiles, mediated by the NRF2 transcription factor. Electrophilic NRF2 activators are used to treat diverse chronic diseases. While the majority of NRF2 activators target C151 of KEAP1, the primary NRF2 repressor, it is unknown how diverse electrophiles favor this particular cellular cysteine. One hypothesis is that the pKa of C151 is lowered by surrounding basic residues, resulting in a higher population of the reactive thiolate. We show that the pKa of C151 is 6.9, providing optimal reactivity at physiological pH, using the fluorogenic, thiol-reactive electrophile monobromobimane. Surprisingly, monobromobimane reacts with C151 much faster than with a small-molecule thiolate. NRF2 activators in clinical use and trials (omaveloxolone, bardoxolone methyl, and sulforaphane) readily compete with monobromobimane for C151. A BTB-monobimane crystal structure shows no specific orientation in the active site after covalent addition. A 4D flexible BTB model based on seven crystal structures was used to dock mBBr and NRF2 activators into the active site to obtain poses of the pre-covalent enzyme-substrate complexes. They reveal an active site around C151 that accommodates structurally diverse activators using largely hydrophobic interactions, with a hydrogen bond orienting their electrophilic carbons within ∼3-5 Å of C151 for a catalytic proximity effect. In addition, our biochemical and docking results suggest a critical catalytic role for hydrogen bonding to the oxygen of an α,β unsaturated carbonyl, which could both increase carbon electrophilicity and stabilize the negative charge in the transition state. Overall, this work suggests enzymatic catalysis is the primary reason that C151 acts as a sensor cysteine for therapeutic, electrophilic NRF2 activators, highly favoring reaction with C151 over other cellular cysteines. The kinetic targeting of electrophiles such as sulforaphane and omaveloxolone to the C151 active site provides an explanation for how electrophilic compounds can be selective pharmacological agents.