Thesis (Ph. D.)--University of Missouri-Columbia, 1998. / Typescript. Vita. Includes bibliographical references (leaves 279-296). Also available on the Internet.

Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 131-146).

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Carboxylesterases are glycosylated general detoxification enzymes belonging to the serine esterase superfamily and play a critical role in the hydrolysis of numerous ester- and amide- containing molecules, including active metabolites, drugs and prodrugs. Three functionally active carboxyleterases have been identified in man (CES1-3), which all show differential tissue expression and critically overlapping, yet specific substrate selectivities. Elucidating the basis of their exact substrate preference would help facilitate the design of clinical prodrugs which are activated by carboxylesterases. Because of their widespread applications, carboxylesterases have attracted much attention in recent years, with CES1 being the most extensively studied human carboxylesterase to date. The work presented here addresses the structure-function relationship of the three human carboxylesterases using a combination of X-ray crystallography, kinetic analysis and biophysical techniques. Recombinant proteins were successfully produced using a mammalian expression system in high yield (5.0 – 84.0 mg/ L cell culture). Analytic ultracentrifugation and size-exclusion chromatography coupled to multi-angle laser light scattering were used to investigate the proteins in solution. These results showed CES1 exists primarily in a trimeric arrangement, whilst CES2 and CES3 are monomeric. Interestingly, atypical mechanisms of substrate inhibition, positive cooperativity and biphasic kinetics were observed for both CES1 and CES2. Three structures of CES1 were solved: wild type, an aglycosylated form and a catalytically inactive form, to 1.48, 1.86 and 2.01 Å respectively. The novel structure of CES2 was solved to 2.04 Å, which revealed that the enzyme forms a strand exchange dimer in contrast to the trimeric CES1. To summarise, this thesis documents a platform that has been generated for the production, characterisation and crystallization of human carboxylesterases. This will aid future structural work for protein ligand binding studies.

Fatty Acid Amide Hydrolase (FAAH), a serine hydrolase family protein, hydrolyzes N-acylethanolamines (NAEs) by cleaving the amide bond linking the acyl group with ethanolamine to produce free fatty acids. Highly conserved ‘Amidase Signature (AS)’ sequence rich in serine, glycine and alanine residues characterize the protein. FAAH plays role in various physiological processes by regulating NAE levels, such as seedling growth, defense response. Understanding of the role of NAEs and FAAH has been however, limited to model plant Arabidopsis. Here, with interest to understand the role of FAAH in modulating NAE composition, tomato was chosen as a model system. Recently, SlFAAH1, an ortholog of AtFAAH1 was identified in tomato and was successfully expressed in prokaryotic expression system. Protein assay with lysate of cells expressing recombinant putative SlFAAH1 showed the ability to hydrolyze a polyunsaturated NAE (NAE20:4). Currently, additional assays are being carried out to determine optimal pH, temp, substrate specificity and associated enzyme kinetics. In parallel, the effect of exogenous NAEs on SlFAAH1 expression levels and during seedling development is being evaluated. Together, this study is expected to not only characterize a protein in tomato but also determine its role in mediating NAE metabolism and seedling development, and further allows for comparison with Arabidopsis and mammalian FAAH to determine its functional conservation.

Protein kinase ASK1 (apoptosis signal-regulating kinase 1) is a member of the mitogen- activated protein kinase kinase kinase (MAP3K) family and plays a crucial role in immune and stress responses. Since the increased activity of ASK1 has been linked to the development of several diseases including cancer, cardiovascular and neurodegenerative diseases, this enzyme is a promising target for therapeutical intervention in these pathologies. The molecule of ASK1 consists of 1374 amino acid residues, but catalytic activity possesses only a kinase domain located approximately in the middle of the molecule. The activity of ASK1 is regulated by interactions with various proteins including the 14-3-3 protein. This protein recognizes a phosphorylated motif around Ser966 at the C-terminus of the catalytic domain of ASK1. This binding interaction inhibits ASK1 through unknown mechanism. ASK1 under stress conditions, such as oxidative stress, is dephosphorylated at Ser966 and the 14-3-3 protein dissociates. This dissociation is then one of the factors that lead to the activation of ASK1. The aim of this diploma thesis was to prepare a complex of the catalytic domain of ASK1 with the 14-3-3 protein for subsequent structural studies. Both proteins were expressed in E. coli cells and successfully purified. In...

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For this pseudoquadreactant enzymatic reaction (Mn2+ is a psuedoreactant), initial velocity patterns were obtained under conditions in which two substrates were maintained saturating while one reactant was varied at several fixed concentrations of the other.

The kinetic mechanism of activation of the NAD-malic enzyme by fumarate and the transition state structure for the oxidation malate for the NAD-malic enzyme reaction have been studied. Fumarate exerts its activating effect by decreasing the off-rate for malate from the E:Mg:malate and E:Mg:NAD:malate complexes. The activation by fumarate results in a decrease in K_imalate and an increase in V/K_malate by about 2-fold, while the maximum velocity remains constant. A discrimination exists between active and activator sites for the binding of dicarboxylic acids. Activation by fumarate is proposed to have physiologic importance in the parasite. The hydride transfer transition state for the NAD-malic enzyme reaction is concerted with respect to solvent isotope sensitive and hydride transfer steps. Two protons are involved in the solvent isotope sensitive step, one with a normal fractionation factor, another with an inverse fractionation factor. A structure for the transition state for hydride transfer in the NAD-malic enzyme reaction is proposed.