title: Raman spectra of interchanging β-lactamase inhibitor intermediates on the millisecond time scale.
abstract: Rapid mix-rapid freeze is a powerful method to study the mechanisms of enzyme-substrate reactions in solution. Here we report a protocol that combines this method with normal (non-resonance) Raman microscopy to enable us to define molecular details of intermediates at early time points. With this combined method, SHV-1, a class A β-lactamase, and tazobactam, a commercially available β-lactamase inhibitor, were rapidly mixed on the millisecond time scale and then were flash-frozen by injection into an isopentane solution surrounded by liquid nitrogen. The "ice" was finally freeze-dried and characterized by Raman microscopy. We found that the reaction is almost complete in solution at 25 ms, giving rise to a major population composed of the trans-enamine intermediate. Between 25 and 500 ms, minor populations of protonated imine are detected that have previously been postulated to precede enamine intermediates. However, within 1 s, the imines are converted entirely to enamines. Interestingly, with this method, we can measure directly the turnover number of SHV-1 and tazobactam. The enzyme is completely inhibited at 1:4 ratio (enzyme:inhibitor) or greater, a number that agrees with the turnover number derived from steady-state kinetic methods. This application, employing non-intensity-enhanced Raman spectroscopy, provides a general and effective route to study the early events in enzyme-substrate reactions.
authors: Department of Chemistry, Case Western Reserve University, Cleveland, Ohio 44106, USA.
authors: Hossein
authors: H
authors: Heidari Torkabadi
authors: Tao
authors: T
authors: Che
authors: Jingjing
authors: J
authors: Shou
authors: Sivaprakash
authors: S
authors: Shanmugam
authors: Michael W
authors: MW
authors: Crowder
authors: Robert A
authors: RA
authors: Bonomo
authors: Marianne
authors: M
authors: Pusztai-Carey
authors: Paul R
authors: PR
authors: Carey
mesh_terms: D004791:Enzyme Inhibitors / Q000737:chemistry*; D013059:Spectrum Analysis, Raman / Q000379:methods*; D065093:beta-Lactamase Inhibitors
chemical_list: D004791:Enzyme Inhibitors; D065093:beta-Lactamase Inhibitors