Reactive architecture profiling with a methyl acyl phosphate electrophile

Abstract

Activity-based protein profiling has facilitated the study of the activity of enzymes in proteomes, inhibitor development, and identification of enzymes that share mechanistic and active-site architectural features. Since methyl acyl phosphate monoesters act as electrostatically selective anionic electrophiles for the covalent modification of nucleophiles that reside adjacent to cationic sites in proteins, we synthesized methyl hex-5-ynoyl phosphate (MHP) to broadly target such protein architectures. After treating the soluble proteome of Paucimonas lemoignei with MHP, biotinylating the resulting acylated proteins using click chemistry, enriching the protein adducts using streptavidin, and analyzing the proteins by LC-MS/MS, a set of 240 enzymes and 132 non-enzyme proteins were identified for a wide spectrum of biological processes and from all 7 enzyme classes. Among those enzymes identified, β-hydroxybutyrate dehydrogenase (PlHBDH) and CTP synthase (E. coli orthologue, EcCTPS) were purified as recombinant enzymes and their rates of inactivation and sites of modification by MHP and methyl acetyl phosphate (MAP) were characterized. MHP reacted more slowly with these proteins than MAP but exhibited greater specificity, despite its lack of multiple binding determinants. Generally, MAP modified more surface residues than MHP. MHP specifically modified Ser 146, Lys 156, and Lys 163 at the active site of PlHBDH. MHP and MAP modified numerous residues of EcCTPS with CTP furnishing the greatest level of protection against MHP- and MAP-dependent modification and inactivation, respectively, followed by ATP and glutamine. Overall, MHP served as an effective probe to identify proteins that are potentially amenable to inhibition by methyl acyl phosphates.