Kinetic Isotope Effects and Transition State Analysis of the Carbapenem-Hydrolysing β-Lactamase KPC-2

The β-lactamase enzymes are the main cause of bacterial resistance towards the most efficient and widely used antibiotics. There is currently a pressing need for discovering novel and potent drugs to counteract this resistance. This thesis represents the first application of the Kinetic Isotope Effects (KIEs) technique to unravel the Transition State (TS) structure of a clinically relevant β-lactamase enzyme. The structural and mechanistic details of the TS provide valuable insight into the development of new β-lactamase inhibitors to combat antibiotic resistance.

Measuring KIEs is an effective and powerful way to access details of an enzymatic TS. By capturing the electronic and structural details of an enzymatic TS in the form of a stable structure (TS analogue), some of the most potent enzyme inhibitors to date have been developed. The implementation of the TS analysis technique for the most clinically relevant β-lactamases then represents a promising lead.

This thesis first focuses on characterising three of the most prevalent βlactamases which have the ability to hydrolyse the “last-resort” carbapenem antibiotics, to assess their viability as targets for the measurement of KIEs. Then, the technical challenges to implement and carry out the competitive dual-label KIE technique for the KPC-2 β-lactamase are addressed. Lastly, the experimental KIEs are determined and used in combination with quantum-mechanical (QM) electronic structure calculations to derive a computational model of the likely TS.

Chapter 2 provides an overview of the KPC-2, OXA-48 and NDM-1 carbapenem-hydrolysing β-lactamases. It also presents the experimental results pertaining the heterologous expression and kinetic characterisation of these β-lactamases with benzylpenicillin. The kinetic data for the hydrolysis reaction of these enzymes was used to assess their suitability as targets for the measurement of KIEs and to find the most optimal reaction conditions for their determination.

Chapter 3 describes the experimental requirements for carrying out the KIE experiments for KPC-2 using benzylpenicillin as substrate. This work involved a methodology for quenching the enzymatic reaction and a chromatographic purification method to isolate the substrate and product from the reaction mixture. Benzylpenicillin substrates carrying heavier stable isotopes in reaction-sensitive positions and remote radioactive labels were prepared following a chemoenzymatic synthetic route. The internal competition of the substrates with the heavier versions during the enzymatic reaction allowed for the expression and calculation of the intrinsic KIEs.

Chapter 4 provides an analysis of the KPC-2 mechanism, highlighting the significance of the intrinsic KIE values in elucidating the details of the TS structure. Additionally, it showcases the computational modelling efforts, integrating QM calculations with the intrinsic KIEs to derive the TS structure.

Finally, a TS model in alignment with the majority of the experimental KIEs was located. This structure represents a late TS occurring during the acylation step of the mechanism, featuring a fully formed acyl bond, a tetrahedral carbon geometry, and an extended β-lactam amide bond. Chapter 5 then discusses how these findings make a significant contribution to the understanding of the enzymatic hydrolysis mechanism of benzylpenicillin by KPC-2. Furthermore, an example is given on how the TS structure can be used as a blueprint for the design of TS analogue structures with potential as inhibitors of this enzyme.