Direct characterization of enzyme-substrate complexes by using electrosonic spray ionization mass spectrometry.

Numerous reports have cited the use of electrospray ionization (ESI) mass spectrometry to characterize intact, multiplycharged proteins and protein complexes. Herein we describe the application of a variant method, electrosonic spray ionization (ESSI), to monitor dynamic changes associated with formation of binary and ternary complexes, such as enzyme–substrate and enzyme–substrate–inhibitor systems. The basis for the experiment is that the gentle ESSI ionization method preserves solution protein and protein complex structures and tags each protein with a charge state that is characteristic of its conformation. In the course of the study, we show that noncovalent solution interactions between the enzyme imidazole-3-glycerol phosphate synthase (IGP synthase) and the substrate/inhibitor are preserved in the gas-phase ions. This enzyme contains two distinct active sites, the glutaminase domain and the synthetase domain located some 30 apart. The observations indicate the capacity of this method to preserve native-like protein

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