Thesis (Ph. D.)--West Virginia University, 2008. / Title from document title page. Document formatted into pages; contains xii, 102 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 92-102).

Thesis (Honors)--College of William and Mary, 2009. / Includes bibliographical references (leaves 53-54). Also available via the World Wide Web.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

α-Isopropylmalate synthase (α-IPMS) is responsible for catalysing the first committed step in leucine biosynthesis. This pathway is found in plants and microorganisms, including pathogenic bacteria such as Mycobacterium tuberculosis and Neisseria meningitidis. α-IPMS catalyses a Claisen condensation reaction between α-ketoisovalerate (KIV) and acetyl coenzyme A (AcCoA) to form the product α-isopropylmalate (IPM). This enzyme undergoes feedback inhibition by the end product of the pathway, leucine. This regulation allows the control of the rate leucine biosynthesis. This project focuses on the α-IPMS enzymes from M. tuberculosis and N. meningitidis (MtuIPMS and NmeIPMS). These α-IPMS enzymes are homodimeric in structure. Each monomer consists of a catalytic domain which comprises of a (β/α)8 barrel fold, two subdomains and a regulatory domain, to which the allosteric binding of the natural inhibitor leucine occurs. The mechanism by which the allosteric binding of leucine leads to a decrease in enzymatic activity is not yet fully understood. Citramalate synthase (CMS) is responsible for catalysing the first committed step of threonine-independent isoleucine biosynthesis. This enzyme is extremely similar to α-IPMS in both the reaction which it catalyses and the catalytic and regulatory domain structure. CMS catalyses a Claisen condensation reaction between pyruvate and AcCoA to produce citramalate (CM). CMS is also feedback inhibited by the end product of its pathway, isoleucine. The similarity between α-IPMS and CMS enzymes resulted in and examination of the inhibitor selectivity of MtuIPMS. Amino acids in the leucine binding site were altered to their counterparts in the isoleucine binding site of the CMS enzyme to see if the selectivity of the leucine binding site could be interchanged. Results from this study show that it is possible to change inhibitor selectivity with a single amino acid substitution. However, changing the selectivity from leucine to isoleucine was unsuccessful. Instead, one of the MtuIPMS variants displayed significantly increased sensitivity to an alternative amino acid, norvaline. The MtuIPMS variants were expressed and purified using immobilised metal affinity chromatography and size-exclusion chromatography. These variants were then kinetically characterised and displayed similar binding affinities and turnover rates for the natural substrates to the wild-type enzyme. As expected changes to the leucine binding pocket had drastic effects on the sensitivity of the enzyme to its natural inhibitor. This work is described in Chapter 2 of this thesis. The mechanism by which the regulatory signal is transferred from the allosteric leucine binding site to the catalytic site in α-IPMS is not fully understood. NmeIPMS variants were created based on preliminary molecular dynamic simulations which indicated that significant changes in residue contacts were associated with leucine binding. Chapter 3 describes studies that explore the effect of single amino acid substitutions of NmeIPMS. The NmeIPMS variants were expressed and purified similarly to MtuIPMS, using immobilised metal affinity chromatography and size-exclusion chromatography. Variants were subsequently characterised via mass spectrometry, differential scanning fluorimetry and kinetic assays. It was found that each variant generated retained sensitivity to leucine but displayed significant differences in the catalytic efficiencies with AcCoA. One of the generated variants also displayed a significant increase in thermal stability. Results are drawn together in Chapter 4 along with future directions of this research. This chapter details knowledge gained into protein structure and allosteric mechanisms in this thesis.

This thesis reports a study of polydentate amino acid complexes of cobalt(III). The complexes prepared during this project have been characterized by a range of techniques, including ¹³C{¹H} and ¹H NMR spectroscopy, UV-visible spectroscopy, infra-red spectroscopy, elemental analysis and single crystal X-ray structure determination. A total of seven single crystal X-ray structure determinations have been performed during these studies. The imino acid polydentate complex, [Co(Aim₂trien)]₂[ZnCl₄], was reduced to the corresponding amino acid complex, [Co(A₂trien)]Cl, where as many as ten diastereoisomers could be formed due to the formation of new stereogenic centres. The crude product of these reactions was a mixture of isomers, according to ¹³C{¹H} NMR data. These isomers were separated using ion-exchange chromatography. The major isomer (I1), a minor isomer (I2a) and a half reduced complex (I4a) from the [Co(A₂trien)]Cl reduction and separation experiment were characterised. The predominant isomers produced were found to have had the proton on the α-carbon atoms positioned on the amine face of each amino acid ligand fragment. To investigate the ratio of the isomers formed by the initial borohydride reduction, an isomerisation study of the major isomer of the [Co(A₂trien)]⁺ complex (I1) was performed. This study hoped to establish the degree to which the distribution of isomers was a result of dynamic equilibrium. Experiments on a small scale showed the initial isomer distribution to be similar to that obtained from the borohydride reduction reaction. However, prolonged exposure to the carbonate buffer (≈ two weeks) resulted in isomers not previously seen. Experiments on a large scale were performed to establish whether the results were consistent. The materials from both the two hour and two week experiments were mixtures of isomers by ¹³C{¹H} NMR spectroscopy and were separated using ion-exchange chromatography. ¹H NMR data of the two hour experiment showed only epimerisation of the amine proton adjacent to the α-carbon atom. Therefore the isomers produced from the isomerisation of I1 have the same configuration of the proton on the α-carbon atoms, which is on the amine face of each amino acid chelate ring. ¹H NMR data from the two week experiment resulted in new isomers not previously seen as both the amine proton and the proton on the α-carbon atom have been epimerised. The polyamine wrapping around the central metal ion may also have changed in some cases. It would appear, from the ¹H NMR data that the methyl group signals of these isomers fall in two distinct clusters; a cluster at δ 1.50-1.65 ppm and a cluster at δ 1.40-1.49 ppm. From these results, and the results of Chapter Two, it has been calculated that there is at least 92% facial selectivity for the amine face of the molecule during the initial borohydride reduction reactions. This may be due to a di-hydrogen bonding interaction between an adjacent amine proton and a hydride of the borohydride, which directs the attack. Following on from this study, a new range of imino and amino acid complexes were synthesised using different tetraamine and pentaamine cobalt(III) complexes. X-ray quality crystals of [Co(Aim₂2,2,3-tet)][ClO₄] and [Co(Aim₂2,3,2-tet)][ClO₄] were obtained and solved with assistance from Dr. Chris Fitchett and Dr. Jennifer Burgess. Borohydride reductions were performed on the [Co(Aim₂2,2,3-tet)]⁺ and [Co(Aim₂2,3,2-tet)]⁺ systems. The products were a mixture of isomers according to 1H and ¹³C{¹H} NMR spectroscopy. The results from the ¹H NMR experiments showed similarity between the [Co(A₂2,3,2-tet)]⁺ and [Co(A₂trien)]⁺ systems, where three major stereoisomers were present in solution. Analogous results for the asymmetric [Co(A₂2,2,3-tet)]⁺ system were also observed. Preliminary attempts have been made to separate these isomers using ion-exchange chromatography.

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This thesis reports a study of polydentate amino acid complexes of cobalt(III). The complexes prepared during this project have been characterized by a range of techniques, including ¹³C{¹H} and ¹H NMR spectroscopy, UV-visible spectroscopy, infra-red spectroscopy, elemental analysis and single crystal X-ray structure determination. A total of seven single crystal X-ray structure determinations have been performed during these studies. The imino acid polydentate complex, [Co(Aim₂trien)]₂[ZnCl₄], was reduced to the corresponding amino acid complex, [Co(A₂trien)]Cl, where as many as ten diastereoisomers could be formed due to the formation of new stereogenic centres. The crude product of these reactions was a mixture of isomers, according to ¹³C{¹H} NMR data. These isomers were separated using ion-exchange chromatography. The major isomer (I1), a minor isomer (I2a) and a half reduced complex (I4a) from the [Co(A₂trien)]Cl reduction and separation experiment were characterised. The predominant isomers produced were found to have had the proton on the α-carbon atoms positioned on the amine face of each amino acid ligand fragment. To investigate the ratio of the isomers formed by the initial borohydride reduction, an isomerisation study of the major isomer of the [Co(A₂trien)]⁺ complex (I1) was performed. This study hoped to establish the degree to which the distribution of isomers was a result of dynamic equilibrium. Experiments on a small scale showed the initial isomer distribution to be similar to that obtained from the borohydride reduction reaction. However, prolonged exposure to the carbonate buffer (≈ two weeks) resulted in isomers not previously seen. Experiments on a large scale were performed to establish whether the results were consistent. The materials from both the two hour and two week experiments were mixtures of isomers by ¹³C{¹H} NMR spectroscopy and were separated using ion-exchange chromatography. ¹H NMR data of the two hour experiment showed only epimerisation of the amine proton adjacent to the α-carbon atom. Therefore the isomers produced from the isomerisation of I1 have the same configuration of the proton on the α-carbon atoms, which is on the amine face of each amino acid chelate ring. ¹H NMR data from the two week experiment resulted in new isomers not previously seen as both the amine proton and the proton on the α-carbon atom have been epimerised. The polyamine wrapping around the central metal ion may also have changed in some cases. It would appear, from the ¹H NMR data that the methyl group signals of these isomers fall in two distinct clusters; a cluster at δ 1.50-1.65 ppm and a cluster at δ 1.40-1.49 ppm. From these results, and the results of Chapter Two, it has been calculated that there is at least 92% facial selectivity for the amine face of the molecule during the initial borohydride reduction reactions. This may be due to a di-hydrogen bonding interaction between an adjacent amine proton and a hydride of the borohydride, which directs the attack. Following on from this study, a new range of imino and amino acid complexes were synthesised using different tetraamine and pentaamine cobalt(III) complexes. X-ray quality crystals of [Co(Aim₂2,2,3-tet)][ClO₄] and [Co(Aim₂2,3,2-tet)][ClO₄] were obtained and solved with assistance from Dr. Chris Fitchett and Dr. Jennifer Burgess. Borohydride reductions were performed on the [Co(Aim₂2,2,3-tet)]⁺ and [Co(Aim₂2,3,2-tet)]⁺ systems. The products were a mixture of isomers according to 1H and ¹³C{¹H} NMR spectroscopy. The results from the ¹H NMR experiments showed similarity between the [Co(A₂2,3,2-tet)]⁺ and [Co(A₂trien)]⁺ systems, where three major stereoisomers were present in solution. Analogous results for the asymmetric [Co(A₂2,2,3-tet)]⁺ system were also observed. Preliminary attempts have been made to separate these isomers using ion-exchange chromatography.

Thesis (Ph. D.)--Washington State University, May 2008. / Includes bibliographical references (p. 88-98).

Thesis (Ph. D.)--University of Wisconsin--Madison, 1965. / Vita. Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.