Beyond enzyme kinetics: Direct determination of mechanisms by stopped-flow mass spectrometry

The development of soft ionization techniques has made mass spectrometry an efficient and essential tool for the determinations of the primary structures of peptides and proteins. Recently the technique has been extended at an explosive rate to noncovalent structures as well as dynamics of protein-protein interactions. We propose here that interfacing mass spectrometry with a stopped-flow mixing device and applying these new techniques of soft ionization to enzymes undergoing catalysis will provide direct access to enzyme mechanisms, both kinetic mechanisms (which describe the comings and goings of substrates, products, and inhibitors) and chemical mechanisms (which describe the order of breaking and making chemical bonds). Transient-state measurements will provide the order of reaction events; steady-state measurements will provide the distribution and therefore the relative energy level of enzyme forms participating in those events; combining transient-state and steady-state measurements is therefore expected to provide sufficient information to construct a free energy diagram of the enzyme-catalyzed reaction. (C) 1997 Elsevier Science Ltd.