Theses and Dissertations at Montana State University (MSU)
Permanent URI for this communityhttps://scholarworks.montana.edu/handle/1/732
Browse
5 results
Search Results
Item Biochemical characterization of the six-transmembrane epithelial antigen of the prostate family of metalloreductases(Montana State University - Bozeman, College of Letters & Science, 2015) Kleven, Mark Daniel; Chairperson, Graduate Committee: C. Martin Lawrence; George H. Gauss was the main author, and Mark D. Kleven, Anoop K. Sendamarai, Mark D. Fleming and C. Martin Lawrence were co-authors of the article, 'The crystal structure of six-transmembrane epithelial antigen of the prostate 4 (Steap4), a ferri/cuprireductase, suggests a novel interdomain flavin-binding site' in the journal 'Journal of biological chemistry' which is contained within this thesis.; Mark D. Fleming and C. Martin Lawrence were co-authors of the article, 'Characterization of a single B-type heme, FAD and metal binding sites in the transmembrane domain of six trans-membrane epithelial antigen of the prostate (Steap) family proteins' submitted to the journal 'Journal of biological chemistry' which is contained within this thesis.Iron and copper are the two most abundant transition metals in humans and are mediators of many essential cellular processes. The entry of these metals into cells require controlled processes, including their reduction prior to uptake. A group of integral membrane enzymes, the six-transmembrane epithelial antigen of the prostate (Steap) family, are able to perform this function. Steap3, in particular, functions as the primary ferric reductase in the transferrin cycle, the dominant mode of erythrocyte iron uptake. How these enzymes perform these functions has remained ill-defined. Here, the biochemical underpinnings of Steap metalloreductase activity have been investigated. To elucidate these mechanisms, expression systems for Steap3 and Steap4 have been developed in bacterial, insect, and human cell lines and purified to varying degrees. By analyzing the truncated cytoplasmic oxidoreductase domain of Steap4, it was found that NADPH is oxidized by transferring a pair of electrons to a flavin. With this truncation, however, flavin only binds weakly and the construct shows no ability to preferentially bind one type of flavin. In contrast, when the full length Steap3 was partially purified, it exhibits high-affinity FAD-binding, indicating that the transmembrane region of the protein contains the major structural features of the FAD binding site. Further, it was found that the cytoplasm-oriented loops between transmembrane helices formed the site. The next cofactor in the electron transport chain is a single b-type heme. Two strictly conserved histidines were identified that coordinate the heme and both are required for heme incorporation. The metal binding site at the extracellular face of the membrane was also characterized. Here, it was found that Steap3 and Steap4 share a conserved high-affinity iron binding site. Additionally, iron and copper both bind with similar affinities to Steap4. Two critical residues of the metal binding site were determined and their predicted proximity to the heme cofactor suggests that the electron is transfer is direct between cofactor and metal. Finally, it was found that Steap's are able to dimerize in the cells, forming homo- and heterodimers Together, the enzymatic mechanism has been characterized in-depth for the first time for these physiologically-significant enzymes.Item Expression and characterization of copper-containing proteins: galactose oxidase and tyrosinase(Montana State University - Bozeman, College of Letters & Science, 2002) Kamlin, Ejan MarieItem Kinetics and mechanism of the reactions of Cu(II)-N,N'-diglycylethylenediamine with triethylenetetramine and ethylenediaminetetraacetate(Montana State University - Bozeman, College of Letters & Science, 1983) Mitchell, Presley KirklandItem Understanding the molecular factors governing inhibitor potency and oxygen activation in copper amine oxidases(Montana State University - Bozeman, College of Letters & Science, 2006) Shepard, Eric Michael; Chairperson, Graduate Committee: David M. DooleyCopper amine oxidases (CuAOs) catalyze the oxidative deamination of primary amines to the corresponding aldehydes, with the concomitant reduction of O2 to H2O2. CuAOs are known to have a multitude of physiological roles, and activity levels are upregulated in several pathological states. With the potential for therapeutic applications, substantial efforts were made to determine the molecular factors governing inhibitor selectivity. As such, several mechanism-based inhibitors were screened against CuAOs from bacterial, yeast, plant, and mammalian sources. The results provided vast insight into the molecular factors governing inhibitor potency in a bacterial (AGAO) and a plant (PSAO) CuAO. This was the defining work in establishing how certain aromatic residues in the substrate channel of AGAO directly control inhibitor potency. Furthermore, this work details the cross-reactivity between CuAOs and monoamine oxidase (MAO) enzymes using the antidepressant MAO-directed inhibitor tranylcypromine, and describes the characterization of a class of compounds which selectively inhibit only CuAOs. In addition to the studies aimed at understanding the molecular factors governing inhibitor binding in CuAOs, this work probes the role of copper during enzymatic reoxidation. Binding and inhibition studies of CuAOs were performed with the monodentate copper ligands cyanide and azide. Studies with cyanide provided the first spectroscopic evidence for cyanohydrin derivitization of TPQ in PSAO and led to the proposal of a detailed mechanism for inhibition of CuAO catalysis by cyanide. Azide studies in PSAO clearly revealed that azide was a competitive inhibitor towards substrate dioxygen, thereby supporting the viability of a redox role for copper. However, azide studies with a yeast CuAO were much more difficult to interpret, as azide was found to significantly inhibit the reaction velocity of both half-reactions. Lastly, temperature jump relaxation measurements of the internal redox equilibrium in AGAO were performed, allowing for the calculation of the electron transfer rate between reduced TPQ and copper(II). The results unequivocally establish that the copper(I)-semiquinone state is a viable catalytic intermediate in the reoxidation reaction of AGAO. Collectively, the results presented in this thesis provide evidence for a redox role of copper during the oxidative half-reaction, although further experimentation is necessary to confirm this.Item Mechanism and inhibition of the phenylethylamine oxidase from Arthrobacter globiformis(Montana State University - Bozeman, College of Letters & Science, 2005) Juda, Gregory Alexander; Chairperson, Graduate Committee: David M. DooleyCopper amine oxidases (CuAOs) catalyze the oxidative deamination of primary amines to the corresponding aldehydes, utilizing O2 as the oxidant with concomitant production of H2O2 and NH3. The discovery that the human vascular adhesion protein (HVAP-1) is a copper amine oxidase has sparked considerable interest in the mechanism and inhibition of these enzymes. With the potential for therapeutic applications, substantial efforts have been made to determine the molecular factors which govern inhibitor sensitivity and selectivity for copper amine oxidases. In order to contribute to this growing body of knowledge, a series of inhibitors were screened for their ability to inhibit enzymatic catalysis in the phenylethylamine oxidase from Arthrobacter globiformis (AGAO). This CuAO was chosen as a model enzyme for these studies due to its ease in expression and purification as a C-terminal Strep-tag II fusion protein. This work describes the kinetic and structural characterization of a new class of highly potent, reversible CuAO inhibitors with inhibition constants in the low nanomolar range. In addition, an investigation of the molecular details of copper amine oxidase catalysis is described; in particular, mechanistic studies of the controversial oxidative half reaction. Studies using the monodentate copper ligand cyanide provided the first spectroscopic evidence for cyanohydrin derivitization of the quinone cofactor in AGAO and led to the proposal of a detailed mechanism for inhibition of CuAO catalysis by cyanide. Lastly, the role of copper during the oxidative half-reaction of amine oxidation in AGAO and Pichia pastoris lysyl oxidase (PPLO) was investigated using the exogenously added copper ligand azide. This work describes the first examination of the effects of azide on both the oxidative and reductive half-reactions of multiple CuAOs. Azide is the first example of a CuAO inhibitor that significantly reduces the catalytic rate for one halfreaction thereby abolishing the kinetic independence of the other half-reaction. Taken collectively, these results show that in the case of some CuAOs it is experimentally impossible to discern the effects of azide on copper mediated enzymatic reoxidation from the inhibition induced during the reductive half-reaction.