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Recombinant giardial arginine deiminase (rADI) was characterized. The enzyme was found to have a specific activity of 12 U (mg protein)-1under at pH 7.4 and 1 mM arginine. The maximum velocity was 14.75 U (mg protein-1) and the KM was 0.167 mM. The specific activity and maximum velocity values are significantly lower than the values reported previously for giardial rADI, while the KM value is quite similar. The optimum pH for the giardial rADI was 6-9, a broad range compared to other arginine deiminases. Recombinant ADI is very specific in its binding specificity, with canavanine (KI 2.4 mM) and ornithine (KI 2.1 mM) being the only substrate analogues giving significant inhibition from the wide variety of analogues tested. None of the analogues could be shown to act as alternative substrates. The contribution of conserved, catalytic and C-terminal residues proposed by previous research towards ADI activity was investigated by site-directed mutagenesis. Mutations of catalytic site residues Asp175, Glu226, His280 and Cys424 decreased the rADI activity to below 2%. Conservative mutations showed significant activity for Asp175 to Glu175 (DE175) and Glu226 to Asp226 (ED226). Site directed mutagenesis on the conserved non-catalytic site Leu46 showed activities below 15%, with the highest activity observed for the mutation to Val46 (LV46), which differs in one CH2 to Leu46 on its side chain. The KM of the mutant LV46 was 3.64 mM while for LA46 (Leu to Ala mutation) was 1.33 mM. Excising 5, 13, 16 amino acids from the C-terminal residues resulted in activity decreasing to 0.8% of the wild type, while excising 54 amino acids caused the rADI to be insoluble. Sequence alignment of Giardia and Dictyostelium suggests a homologous area within the C-terminal extension. Site directed mutagenesis on the Tyr567 residue in this region resulted in a decrease in activity, with the highest activity observed for a Tyr to Phe mutation. Studies using specific cysteine protease inhibitors demonstrated partial protection against proteolysis of ADI against giardial proteases. Degradation of ADI by giardial proteases was more rapid at pH 6 than at pH 7.4.

Graduation date: 1997

Human T-cell lymphotropic virus type I (HTLV-I) is a retrovirus that is the causative agent of the fatal disease adult T-cell leukemia (ATL). HTLV-I silently infects over twenty million people worldwide; up to ten percent of these will develop ATL in their lifetime. There are currently no effective treatments for this disease. HTLV-I expresses its genome as polypeptides that must be processed in order to produce infectious virions. Like other retroviruses, HTLV-I encodes an aspartic acid protease to process these polypeptides into mature form. Because the protease is essential in the virus life cycle, it is an attractive target for the treatment of HTLV-I-induced ATL. The present work examines the structure and function of HTLV-I protease. A theoretical structure of the protease is presented, and the function of the C-terminal extension is considered. In order to determine which residues are involved in binding substrate, two experiments were performed: first, several residues were mutated to the corresponding residues in HIV-1 protease to determine whether HTLV-I protease can be made to process an HIV-1 protease substrate; second, an alanine scan was performed to knock out individual residues to assess their importance in binding substrate. This work builds knowledge of the structure and function of HTLV-I protease. By understanding which residues play a role in binding substrate and by developing a clearer picture of the structure of the protease, it will be possible to develop specific inhibitors for HTLV-I protease.

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 76-85).

Current enzyme kinetic equations are inadequate for modelling enzymatic reactor systems because they fail to take into account the interactions between that various process parameters. They also are unable to predict reaction rates in complex solute systems. A quasi-native kinetic model was developed that predicts enzyme activity by examining the effect of solute addition on the overall protein structure. The theory was tested using the enzyme urease (urea aminohydrolase EC 3.5.1.5). / The quasi-native model was found to accurately predict both the activation and inhibition phenomena observed with urease and could also predict enzymatic activity in complex solute systems. The quasi-native isomerization constant was shown to be a function of hydrophobic effects characterized by the Sechenov theory and electrostatic effects characterized by the DeBye-Huckel theory. The Sechenov constant was found to be independent of temperature and pH. / The urease denaturation rate constant displayed a response to solute addition similar to that observed with the quasi-native isomerization equilibrium constant. However, the effect of pH on urease kinetics was a complex function of the ionization of active-site ligands and enzyme surface charge interactions.

The main objective of this research is to advance our understanding of the biochemical properties and the structure-function relationships of the chloroplast glycerol-3-phosphate acyltransferases in plants. De novo synthesised fatty acyl chains are diverted into the prokaryotic pathway of plant lipid biosynthesis by a soluble glycerol-3-phosphate acyltransferase (GPAT [EC. 2.3.1.15]) in the chloroplast. GPAT catalyses acylation at the sn- 1 position of sn-glycerol-3-phosphate to form lysophosphatidic acid. Recombinant GPAT from squash and Arabidopsis were overproduced in Escherichia coli, purified to about 23- fold and 90% pure enzyme using a procedure developed in this study. Antibodies were raised in rabbits against these denatured recombinant GPAT preparations and four peptide antigens, and preliminary experiments were performed to test their suitability for use in Western blotting. In collaboration with the University of Sheffield, squash GPAT was successfully crystallised, isomorphous heavy metal derivatives prepared and the complete 3-dimensional structure of the protein at 2.3 Angstrom resolution determined. The cloning, functional expression and characterisation of a novel GPAT from oil palm, 'domainswap' chimeric recombinant proteins of Arabidopsis and squash GPAT, and spinach and squash GPAT respectively, and the influence of the N-terminal domain and amino acid substitutions in the C-terminal domain of the squash GPAT, was described. By determining the apparent kinetic constants for acyl-ACP substrates of most of the enzymes and by in vitro assays using mixtures of two acyl-ACP substrates under physiologically relevant conditions, it was found that their substrate selectivities could be dramatically altered. The development of ribozyme- technology as a molecular tool to down-regulate the gene expression of one out of multiple GPATs, was initiated. The strategy would allow for a phenotypic indication of ribozyme- efficacy in vivo and may help further contribute to the role of glycerol-3-phosphate acyltransferase in processes determining the phenomenon of chilling-sensitivity of plants.

Thesis (M.S.)--Brandeis University, 2009. / Title from PDF title page (viewed on June 29, 2009). Includes bibliographical references.

Thesis (Ph.D.) in Forest Resources--University of Maine, 2008. / Includes vita. Includes bibliographical references (leaves 110-129).

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