Three aspects of chemical and biochemical reactions were investigated. 1. The relative reactivities of pyrophosphate (phosphorus(V)) and pyro-di-H-phosphonate (phosphorus(III)) and its derivatives have been analysed at various pHs. The hydrolysis rate of pyro-di-H-phosphonate (PP(III)) was found to be higher than pyrophosphate at all pHs. Using ITC and NMR, pyrophosphate showed metal-ion complexing abilities whereas pyro-di-H-phosphonate showed weak or no complexing to metal-ions, although the rate of hydrolysis at pH 7 slightly increased compared to the spontaneous hydrolysis of PP(III). The enzymatic hydrolysis of pyrophosphate, which is thought to occur via MgPP(V)2-, occurs efficiently and is close to being diffusion controlled. Pyro-di-H-phosphonate on the other hand does not act as a substrate or as an inhibitor of pyrophosphatase. 2. Dichloromethane (DCM) is an alkylating agent for pyridine, producing methylene bis-pyridinium dication (MDP) upon refluxing the solution. The kinetics and mechanism of hydrolysis of methylene bis-pyridinium dication have been studied. Below pH 7 MDP is extremely stable and hydrolysis is first-order in hydroxide-ion. Above pH 9 an unusual intermediate is formed on hydrolysis which has a chromophore at 366 nm in water and its formation is second-order in hydroxide-ion. The carbon acidity of the central methylene group was also investigated kinetically using H/D exchange and the pKa was surprisingly high at 21.2 at 25oC (I = 1.0 M). 3. Isothermal titration calorimetry (ITC) is a technique mainly used by biochemists to obtain a range of physical and thermodynamic properties of a reaction. Analysing the data can become difficult when investigating complex reactions involving more than one step, for instance metal-ions binding to an enzyme. In this work models have been developed to simulate sequential reactions. These were used to simulate experimental ITC data for metal-ions: Zn2+, Co2+ and Cd2+ complexing to the active sites of BcII, a metallo β-lactamase responsible for antibiotic resistance, providing additional information on the mechanism by which this enzyme acts to deactivate β-lactam antibiotics. The simulations suggest that BcII has two very similar binding affinities to metal-ions which are filled sequentially.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:638113 |
| Date | January 2014 |
| Creators | Mistry, Dharmit |
| Publisher | University of Huddersfield |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://eprints.hud.ac.uk/id/eprint/23444/ |
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