Mechanism-based inhibitors of class A beta-lactamases, such as sulbactam, undergo a complex series of chemical reactions in the enzyme active site. Formation of a trans-enamine acyl-enzyme via a hydrolysis-prone imine is responsible for transient inhibition of the enzyme. Although the imine to enamine tautomerization is crucial to inhibition of the enzyme, there are no experimental data to suggest how this chemical transformation is catalyzed in the active site. In this report, we show that E166 acts as a general base to promote the imine to enamine tautomerization.