| To understand the biosensing mechanism of the recently developed fluorescein-labeled (E166Cf) and badan labeled (E166Cb) beta-Lactamase biosensors, the kinetics and structural basis of the binding reactions between the biosensors and beta-lactam antibiotics were investigated by electrospray ionization mass spectrometry (ESI-MS).;Complementary time-dependent mass spectrometric and fluorometric studies show that the time-resolved fluorescence profile is correlated with the concentration-time profile of the covalently bound acyl enzyme intermediate (ES*) monitored by ESI-MS, unambiguously indicating that the fluorescence emission enhancement is due to substrate binding.;Detailed mass spectrometric kinetics studies revealed that the covalently bound enzyme-substrate complex (ES*) formed from the biosensors is very stable, so that the fluorescence signal emitted by it could be prolonged steady. In addition, the overall binding efficiency, as indicated by the ratio of kinetic parameters k2/Kd, is not significantly impaired by the introduction of the bulky fluorophore into the omega-loop of the beta-lactamases, probably attributed to that the flexible nature of the omega loop and the fluorophore-induced increase in flexibility of the active site binding pocket relieve the steric hindrance exerted by the fluorophore. Furthermore, the incorporation of badan was found to enhance the overall binding efficiency of the enzyme by ∼10-fold, most likely due to that the badan destabilizes the active site and thus alleviates the steric hindrance to a greater extend.;ESI-MS based hydrogen-deuterium (H-D) exchange studies show that for both E166Cf and E166Cb, the fluorophore may be initially oriented towards and close to the active binding site, and induce destabilizing effect to this confined region. Upon substrate binding, the fluorophore has to displace away from the active site in order to prevent the spatial clash with the incoming substrate, thus the destabilizing effects initially exerted by fluorophore to this region are withdrawn. This "spatial displacement" event is likely the source of the fluorescence changes observed upon substrate binding.;Due to discrepancies in reported results obtained by ESI-MS under acidic, denaturating conditions and other physical techniques such as x-ray crystallography and UV spectroscopy, the inhibition mechanism of tazobactam (MW=300 Da) towards beta-lactamases was re-investigated by ESI-MS but under near-physiological conditions. Unlike previous ESI-MS studies, a covalently bound enzyme-inhibitor complex (E-I complex) with relative molar mass of [M+300] Da was observed, which is consistent with the formation of a trans-enamine species as suggested by X-ray crystallography and spectroscopic methods. In addition, our results show, for the first time, that the E-I complex formed from the Staphylococcus aureus PC-1 beta-lactamase and tazobactam dissociates further to form an inactive dehydrated enzyme. Based on the results obtained by protease digestion and tandem mass spectrometry, this dehydrated enzyme is proposed to be an alkene-like species formed from dissociation of the trans-enamine species. For this inhibition mechanism, the role of the inhibitor is initial binding to the active site of the enzyme, followed by triggering of a chemical reaction (or reactions) that result in the formation of an inactivated form of beta-lactamase. |
