<p>  </p> <p>Sulfonation is a widespread biological reaction catalyzed by a supergene family of enzymes called sulfotransferases (SULTs). SULTs utilize 3’-phosphoadenosine-5’-phospho-sulfate (PAPS) as the universal sulfonate donor to conjugate with a diverse range of endo- and xenobiotic substrates, including neurotransmitters, hormones, and drugs resulting in altering their biological activity. This reaction serves as a major detoxification pathway as conjugation with a sulfonate group renders substrates more hydrophilic and facilitates excretion. Therefore, this process is responsible for reducing the bioavailability of some drugs. In some cases, sulfo-conjugation causes the bio-activation of pro-mutagens and pro-carcinogens, leading to SULTs being risk factors in some cancers. Despite the biological relevance, understanding of this family of enzymes is still scarce. One SULT member that is the focus of the studies described herein is human sulfotransferase 2B1b (SULT2B1b), which had been identified as a potential drug target in prostate cancer. However, the inconsistency in reported kinetic data obtained using radiolabeled assays and the lack of robust assays have become significant limitations for SULT2B1b-targeted drug discovery studies. A label-free assay was developed to bridge this knowledge gap that directly quantifies SULT2B1b sulfonated products. This novel assay utilized high-throughput technology based on Desorption Electrospray Ionization Mass Spectrometry (DESI-MS). Results obtained from the DESI-MS-based assay were compared with those from a fluorometric, coupled-enzyme assay already developed in the Mesecar lab. Both methods provided consistent kinetic data for the reaction of SULT2B1b. Therefore, this novel assay is promising for the application of drug discovery efforts aiming at identifying SULT2B1b inhibitors. The other SULT member studied and described herein is human sulfotransferase 1A1 (SULT1A1), one of humans' most vital detoxifying and drug-metabolizing SULT isoforms that can also be a potential drug target in some cancers. The detailed kinetic mechanism of SULT1A1 was elucidated using steady-state kinetic, product inhibition, dead-end inhibition, and X-crystallographic studies. to gain insights into the role of this enzyme in detoxification, drug metabolism, and the development of inhibitors.</p>

β-lactam antibiotics have been the most widely used drug of choice to combat infectious disease caused by bacteria. Unfortunately, their effectiveness is drastically threatened by bacterial β-lactamases. β-lactamases is responsible for the resistance to most antibiotic drugs. For decades, β-lactam β-lactamases inhibitors have been used to reduce bacterial resistance; however, in this study 1,2,4-oxadiazolidinone derivatives as a non-β-lactam β-lactamases inhibitor against TEM-1 and P99 β-lactamases. The significance of oxadiazolidinone is the prominent five-membered ring scaffold in its structure, which is configurationally stable and present in other biologically active compounds such as linezolid and avibactam. Oxadiazolidinones were synthesized by treating nitrones with isocyanates. The synthesized compounds were characterized using 1H and 13C NMR, GC-MS, and FTIR. Afterward, they were tested using Nitrocefin as substrate to determine their effectiveness against TEM-1 and P99 serine β-lactamase. Compound 2a-2c, and 3 showed inhibition ranging from 12-38%.

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Intrinsic structural and conformational mechanisms regulate the functional specificity of the coagulation system. The study of these structure-function relationships is important for understanding the strategies used in the management of clinical thrombosis. Previous studies have shown that the central enzyme in clotting, thrombin, is sequestered inside of a clot, and protected from the natural downregulator antithrombin (AT). This is problematic for anticoagulants like heparin which depend on AT. Subsequently, it was found that the key upstream propagator of thrombin, the prothrombinase enzyme complex, is also resistant to the AT-heparin. Our data show that further upstream of prothrombinase, the intrinsic tenase is only moderately protected, while there is no protection at the level of the initiator complex, extrinsic tenase. This protection phenomenon possibly reflects steric and allosteric mechanisms that ensure maximal activation of the coagulation system once a threshold stimulus is achieved. These mechanisms likely evolved as a result of conformational rearrangement, as evidenced by the proteolytic activation of thrombin activity following proteolysis of prothrombin. Indeed, subtle differences in the structural interaction of ligands with the active site can lead to substantial differences in enzyme activity. The binding of rivaroxaban and apixaban to factor Xa is nearly identical; both interact with the active site with comparable affinity. Despite this, a 3-fold faster rate of the rivaroxaban on-rate yields significantly greater prolongation of the prothrombin time (PT) and activated partial thromboplastin time (aPTT), global tests of coagulation. These small differences in ligand interaction also have allosteric consequences. Structural differences between the direct thrombin inhibitors dabigatran and argatroban yield divergent exosite-mediated thrombin binding to physiologic ligands like yA-fibrin, y'-fibrin, factor Va, and factor VIII, interactions that govern clot-mediated protection from AT inhibition, and the various functions of thrombin. These divergent effects were robust and ligand-dependent, suggesting conserved energetic scaffolds within the thrombin molecule that govern allosteric changes throughout the molecule. Because proteolysis of prothrombin yields significant allosteric and structural rearrangement that capacitates the active site for substrate recognition amd catalytic ability, we investigated the role of Ser195, a key residue in the thrombin catalytic triad in also regulating thrombin allostery. Site directed mutagenesis of Ser195 to Ala yielded a significant increase in the flexibility of the entire thrombin molecule, as evidenced by increased potency of dabigatran and argatroban in terms of their capacity to modulate exosite binding through the active site, and increased interexosite cooperative and competitive allostery. Together, these studies represent an advance in our understanding of the consequences of both small molecule ligation of coagulation proteases, as well as the consequences of subtle structural modification for overall allosteric function. / Thesis / Doctor of Philosophy (PhD)

For systems biology, it is important to describe the kinetic and thermodynamic properties of enzyme-catalyzed reactions and reaction cascades quantitatively under conditions prevailing in the cytoplasm. While in part I kinetic models based on irreversible thermodynamics were tested, here in part II, the influence of the presumably most important cytosolic factors was investigated using two glycolytic reactions (i.e., the phosphoglucose isomerase reaction (PGI) with a uni-uni-mechanism and the enolase reaction with an uni-bi-mechanism) as examples. Crowding by macromolecules was simulated using polyethylene glycol (PEG) and bovine serum albumin (BSA). The reactions were monitored calorimetrically and the equilibrium concentrations were evaluated using the equation of state ePC-SAFT. The pH and the crowding agents had the greatest influence on the reaction enthalpy change. Two kinetic models based on irreversible thermodynamics (i.e., single parameter flux-force and two-parameter Noor model) were applied to investigate the influence of cytosolic conditions. The flux-force model describes the influence of cytosolic conditions on reaction kinetics best. Concentrations of magnesium ions and crowding agents had the greatest influence, while temperature and pH-value had a medium influence on the kinetic parameters. With this contribution, we show that the interplay of thermodynamic modeling and calorimetric process monitoring allows a fast and reliable quantification of the influence of cytosolic conditions on kinetic and thermodynamic parameters.

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