The design of halopyridine-based activity-based probes and mechanistic studies of succinylarginine dihydrolase
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An important design aspect of covalent inactivators is the balance between reactivity, or reversibility of reaction, with nucleophiles in solution and reactivity with nucleophiles at a targeted protein site. We previously identified 4-halopyridines as fragment-sized covalent inactivators of the enzyme dimethylarginine dimethylaminohydrolase (DDAH). Binding of these inactivators stabilizes the more reactive pyridinium form while the less reactive neutral form predominates in solution. Herein, we demonstrate that simple 2- and 4-chloropyridines are extensible as covalent modifiers of other proteins within the E. coli proteome, that halogen positioning can impart target selectivity, and that the targets include a subset of Cys-containing purine binding sites. As one specific example, inosine-5’-monophosphate dehydrogenase is shown to be labeled by a 2-chloropyridine at a catalytic Cys305 residue within the inosine binding site. These results indicate that a simple 2- or 4-chloropyridine core can have wider application as a warhead for incorporation into covalent inhibitors of proteins with diverse function. N-succinylarginine dihydrolase (AstB), like DDAH and arginine deiminase, is part of the amidinotransferase (AT) superfamily. AstB shows conservation of the catalytic residues and carries out a similar type of reaction as other hydrolases in the AT superfamily. Herein, we report the mechanistic studies of AstB and provide insights into how this enzyme performs its dihydrolase activity instead of a “mono” hydrolase reaction, which is more prevalent in this superfamily.