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Enzymes are involved in nearly all biological processes, catalyzing reactions with exceptional selectivity and efficiency. Knowledge of reaction mechanisms, whereby a substrate is converted to product through one or more intermediates, is essential for unravelling an enzyme’s role in metabolism. The most used analytical methods for the study of enzyme function include UV–visible fluorescence spectroscopy, X-ray crystallography, two-dimensional nuclear magnetic resonance, and isothermal calorimetry. However, these methods have several limitations that need to be overcome for the detection of transiently populated intermediate states. Recently, mass spectrometry has proven to be an efficient and sensitive method for studying the structural changes occurring at both the protein and substrate level during enzymatic turnover. This chapter will focus on how mass spectrometry has been successfully applied to several unique systems for the elucidation of enzyme reaction mechanisms, kinetic isotope effects, binding constants and catalysis-linked dynamics. |
