Raman spectra of interchanging β-lactamase inhibitor intermediates on the millisecond time scale

Hossein Heidari Torkabadi, Tao Che, Jingjing Shou, Sivaprakash Shanmugam, Michael W. Crowder, Robert A. Bonomo, Marianne Pusztai-Carey, Paul R. Carey

Research output: Contribution to journalArticlepeer-review

11 Scopus citations


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.

Original languageEnglish
Pages (from-to)2895-2898
Number of pages4
JournalJournal of the American Chemical Society
Issue number8
StatePublished - Feb 27 2013


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