NMR spectroscopy studies of enzyme function and inhibition

The work described in this Thesis focuses on the application of NMR spectroscopy methods in understanding the function and inhibition of two protein systems; these are γ-butyrobetaine hydroxylase (BBOX) and the bacterial potassium ion efflux (Kef) system. BBOX belongs to the super family of enzymes called the 2- oxoglutarate (2OG) and FeII dependent oxygenase and is involved in the biosynthesis of L-carnitine in humans and other prokaryotes. BBOX is a current drug target for the treatment of myocardial infarction. Kef is a ligandgated system that protects bacteria from toxic electrophilic species. Kef is inhibited by the binding of cytoplasmic glutathione (GSH) to KTN (K+ transport and nucleotide) binding domains and activated by glutathione-S-conjugates (GS-X). Since bacterial Kef activation during electrophilic exposure is a critical determinant of their survival, perturbation of Kef activity is potentially a novel target for the development of antibiotic drugs.
1H NMR direct ligand-observation was employed to study the binding interaction of the natural substrate γ- butyrobetaine (GBB) and co-substrate 2OG with BBOX. A 1H NMR-based dual-reporter ligand displacement method was developed to assess the nature of inhibitor binding to BBOX i.e to determine whether an inhibitor competes with GBB or 2OG or both. The method was exemplified with a set of isoquinoline-based inhibitors; the results reveal 'cystallographically unexpected' structure-activity relationship with some inhibitors competing 2OG only and some competing both 2OG and GBB. Using 1H NMR spectroscopy, a simple and efficient BBOX inhibition assay was developed for inhibitor IC50 measurement. Similarly, 1H NMR-based assays were applied to demonstrate that the cation-π interaction between the substrates and aromatic cage residues of BBOX play a critical role in BBOX substrate recognition.
1H NMR spectroscopy was applied to show that in the absence of a 2OG oxygenase, ascorbate in the assay mixture is slowly degraded by the dissolved oxygen to yield H2O2 which simultaneously leads to 2OG breakdown into succinate. It is proposed that in the assays of 2OG oxygenases, the apparent increase in the level of "uncoupled" 2OG turnover with ascorbate over time could possibly be due to the artifacts of the ascorbate induced-2OG breakdown instead of being due to enzyme catalysis. Other reducing agents were also found to oxidise identically by the dissolved oxygen as ascorbate in the mixture and result in 2OG breakdown.
In the Kef system, 1H NMR direct ligand-observation was applied to investigate the influence of each functional group of the Kef activating ligand glutathione-S-N-tertiary butylsuccinimide on its binding interaction (KD 0.4 μM) with Kef-QCTD (Q-linker carboxy terminal domain; a KTN domain) from Shewanella denitrificans (sd) with the aim of developing novel non-peptidic ligands (antibacterial agents) of Kef. In addition, 19F NMR was employed to develop an efficient ligand-observed binding assay for Kef that was used for ligand screening as well as measuring their binding dissociation constant value from a single NMR spectrum. Finally, a 1H NMR technique was applied to confirm that the electron density found in the nucleotide binding pocket in the crystal structure of apo-sdKef-QCTD is unambiguously an AMP molecule that is naturally bound to the protein and has a role in stabilising the dimeric form of KTN domains (Kef proteins).

Advisor: Claridge, T