Perspectives for the Study of ¹⁸O Isotope Effects of Enzymatic Phosphoryl Transfer Reactions by ESI-Orbitrap MS

Isotope effects play an important role in investigating enzymatic phosphoryl transfer reactions, which are central in biochemical pathways and environmental metabolism. However, resolving different isotope effects for mechanistic insights into phosphoryl transfer reactions with stable isotope analysis at natural abundance remains challenging. Current analytical methods primarily allow 18O/16O ratio measurements of phosphate and preclude efficient quantification at different reaction extents. Moreover, various oxygen bond changes make mechanistic interpretation of observable O isotope fractionation particularly difficult. Here, we review recent advances in electrospray ionization Orbitrap mass spectrometry for O isotope analysis in phosphate and organophosphates, and the state-of-knowledge on position-specific isotope effects in phosphoryl transfer reactions. Using a kinetic toy model, we illustrate that further development of 18O/16O ratio analysis with Orbitrap MS should include quantification of O isotope fractionation in organophosphate substrates and alcohol leaving groups to evaluate nucleophilic attack and leaving group departure isotope effects.