Reactive architecture profiling with a methyl acyl phosphate electrophile.

Area of Science:

• Biochemistry
• Chemical Biology
• Proteomics

Background:

• Activity-based protein profiling (ABPP) is crucial for studying enzyme activity and inhibitor development.
• Methyl acyl phosphate monoesters are selective electrophiles for modifying nucleophiles near cationic sites in proteins.

Purpose of the Study:

• To synthesize and utilize methyl hex-5-ynoyl phosphate (MHP) as a chemical probe for broad protein targeting.
• To identify proteins amenable to inhibition by methyl acyl phosphates.

Main Methods:

• Activity-based protein profiling using MHP on the soluble proteome of Paucimonas lemoignei.
• Biotinylation via click chemistry, enrichment using streptavidin, and LC-MS/MS analysis.
• Characterization of MHP and methyl acetyl phosphate (MAP) inactivation and modification sites on purified recombinant β-hydroxybutyrate dehydrogenase (PlHBDH) and CTP synthase (EcCTPS).

Main Results:

• Identification of 240 enzymes and 132 non-enzyme proteins modified by MHP.
• MHP demonstrated slower reaction rates but greater specificity compared to MAP.
• MHP specifically modified active site residues Ser 146, Lys 156, and Lys 163 in PlHBDH.
• CTP, ATP, and glutamine protected EcCTPS from MHP and MAP modification.

Conclusions:

• MHP is an effective chemical probe for identifying proteins potentially inhibited by methyl acyl phosphates.
• The study expands the understanding of protein modification by electrophilic probes.
• MHP's specificity offers advantages for targeted protein profiling and inhibitor design.