Exploring the catalytic mechanism of ATPase at the molecular level by tandem mass spectrometry

Abstract

The nucleotide adenosine-5′-triphosphate (ATP) is the coenzyme selected by nature to provide energy for its cellular processes through the ATP hydrolysis reaction. Although the crystal structures and the general working principles of numerous ATP hydrolases (ATPases) are generally known, this omnipresent ATP conversion reaction is not fully understood at the level of local interactions. Questions such as “How does the peptide environment of the active sites of ATPases affect their association with ATP and the consecutive reaction of ATP?” and “Why is the conversion of ATP to ADP preferred over other reactions at the active site?” await detailed answers at the molecular level. Here, tandem mass spectrometry (MS) based techniques are applied to answer these questions. Gas phase studies indicate that the conversion of ATP to ADP is a charge state driven process of which the behaviour varies dramatically with subtle changes in the ATP binding peptide. Of the peptides and peptide mimics studied, only the Ac-Arg-NH₂ form of arginine actively regulates the hydrolysis of ATP, which proceeds through the sequential release of the ADP $•$ peptide complex and ADP. Relative ion activation studies of the fragmentation patterns of the ATP $•$ Ac-Arg-NH₂ complex show that phosphate bond dissociation is preferred over breakage of the non-covalent bond between ATP and the peptide mimic, which coincidentally agrees with the behaviour of catalysed ATP hydrolysis reaction in solution.