ATP-dependent phosphoenolpyruvate carboxykinase (E. C. 4.1.1.49; PCK) is an enzyme that catalyses the reversible conversion of oxaloacetate and ATP into phosphoenolpyruvate, ADP and CO2. PCK is made up of about 500 to 600 amino acid residues and is divided into two roughly equal domains. Upon binding of substrates, the two domains of PCK move towards each other. PCK is well known for its role in gluconeogenesis but in some species, it can have an anaplerotic role. In other species, PCK is important for metabolic steps involved in fermentation.<p>Presented in this thesis are five solved crystal structures of the ATP-dependent form of PCK. Three of the PCK crystal structures determined were from <i>Escherichia coli</i>; one was a complex of ATP, Mg2+ and CO2, the second structure was an ATP, Mg2+, Mn2+, CO2 and oxaloacetate complex and, the third <i>E. coli</i> structure was a Lys213Ser mutant complexed with ATP, Mg2+and Mn2+. Two <i>Anaerobiospirillum succiniciproducens</i> PCK crystal structures were also solved; one was in the native form and the other was an ATP-Mg2+-Mn2+-oxalate complex. <p>In the <i>E. coli</i>-PCK-ATP-Mg2+-CO2 crystal complex structure, the observed location of CO2 was in agreement with a previously determined <i>E. coli</i> PCK-CO2 crystal structure, which incorporated CO2 into the structure by a different technique. The findings from the <i>E. coli</i> PCK-ATP-Mg2+-CO2 crystal structure allowed the reaction mechanism presented in this work to be proposed.<p>The PCK-ATP-Mg2+-Mn2+-CO2-oxaloacetate structure is the first structure where oxaloacetate is observed bound to PCK. Surprisingly, the observed location of oxaloacetate in this structure is 5 Angstroms away from its expected position near Mn2+. Oxaloacetate is weakly bound to a non-catalytic region of the enzyme. It is proposed that when the domains of PCK move towards each other upon binding nucleotide, oxaloacetate experiences steric crowding which results in it being pushed towards the active site to react. <p>Previous kinetic studies on the <i>E. coli</i> PCK mutant Lys213Ser have determined that Mn2+ is unexpectedly inhibitory. A crystal structure of K213S-PCK-ATP-Mg2+-Mn2+ demonstrates that Mn2+ is tetrahedrally coordinated in the active site, not octahedrally as occurred in other structures. By having Mn2+ in the tetrahedral coordination state, substrate binding in the active site of PCK is altered in a way that does not allow catalysis to occur.<p>The two crystal structures of <i>A. succiniciproducens</i> PCK were useful in quantifying the substrate-induced domain movement. A surface active site lid made up of residues 385 to 405 that had never been observed in any of the previous PCK crystal structures was observed in the <i>A. succiniciproducens</i> PCK-ATP-Mg2+-Mn2+-oxalate crystal structure. Mutational studies of this lid have shown it to be essential for the function of PCK; however, its exact function is not certain. It has been proposed that the lid has multiple functions. One is to sequester the substrates from bulk solvent. Another function may be to assist in domain closure. The third function may be to assist in the proper positioning of substrates in the active site.

ATP-dependent phosphoenolpyruvate carboxykinase (E. C. 4.1.1.49; PCK) is an enzyme that catalyses the reversible conversion of oxaloacetate and ATP into phosphoenolpyruvate, ADP and CO2. PCK is made up of about 500 to 600 amino acid residues and is divided into two roughly equal domains. Upon binding of substrates, the two domains of PCK move towards each other. PCK is well known for its role in gluconeogenesis but in some species, it can have an anaplerotic role. In other species, PCK is important for metabolic steps involved in fermentation.<p>Presented in this thesis are five solved crystal structures of the ATP-dependent form of PCK. Three of the PCK crystal structures determined were from <i>Escherichia coli</i>; one was a complex of ATP, Mg2+ and CO2, the second structure was an ATP, Mg2+, Mn2+, CO2 and oxaloacetate complex and, the third <i>E. coli</i> structure was a Lys213Ser mutant complexed with ATP, Mg2+and Mn2+. Two <i>Anaerobiospirillum succiniciproducens</i> PCK crystal structures were also solved; one was in the native form and the other was an ATP-Mg2+-Mn2+-oxalate complex. <p>In the <i>E. coli</i>-PCK-ATP-Mg2+-CO2 crystal complex structure, the observed location of CO2 was in agreement with a previously determined <i>E. coli</i> PCK-CO2 crystal structure, which incorporated CO2 into the structure by a different technique. The findings from the <i>E. coli</i> PCK-ATP-Mg2+-CO2 crystal structure allowed the reaction mechanism presented in this work to be proposed.<p>The PCK-ATP-Mg2+-Mn2+-CO2-oxaloacetate structure is the first structure where oxaloacetate is observed bound to PCK. Surprisingly, the observed location of oxaloacetate in this structure is 5 Angstroms away from its expected position near Mn2+. Oxaloacetate is weakly bound to a non-catalytic region of the enzyme. It is proposed that when the domains of PCK move towards each other upon binding nucleotide, oxaloacetate experiences steric crowding which results in it being pushed towards the active site to react. <p>Previous kinetic studies on the <i>E. coli</i> PCK mutant Lys213Ser have determined that Mn2+ is unexpectedly inhibitory. A crystal structure of K213S-PCK-ATP-Mg2+-Mn2+ demonstrates that Mn2+ is tetrahedrally coordinated in the active site, not octahedrally as occurred in other structures. By having Mn2+ in the tetrahedral coordination state, substrate binding in the active site of PCK is altered in a way that does not allow catalysis to occur.<p>The two crystal structures of <i>A. succiniciproducens</i> PCK were useful in quantifying the substrate-induced domain movement. A surface active site lid made up of residues 385 to 405 that had never been observed in any of the previous PCK crystal structures was observed in the <i>A. succiniciproducens</i> PCK-ATP-Mg2+-Mn2+-oxalate crystal structure. Mutational studies of this lid have shown it to be essential for the function of PCK; however, its exact function is not certain. It has been proposed that the lid has multiple functions. One is to sequester the substrates from bulk solvent. Another function may be to assist in domain closure. The third function may be to assist in the proper positioning of substrates in the active site.

From opportunistic protist Cryptosporidium parvum we identified and functionally assayed a fatty acyl-CoA-binding protein (ACBP) gene. The CpACBP1 gene encodes a protein of 268 aa that is three times larger than typical ~10 KD ACBPs of humans and animals. Sequence analysis indicated that the CpACBP1 protein consists of an N-terminal ACBP domain (approximately 90 aa) and a C-terminal ankyrin repeat sequence (approximately 170 aa). The entire CpACBP1 open reading fragment (ORF) was engineered into a maltose-binding protein fusion system and expressed as a recombinant protein for functional analysis. Acyl-CoA-binding assays clearly revealed that the preferred binding substrate for CpACBP1 is palmitoyl-CoA. RT-PCR, Western blotting and immunolabelling analyses clearly showed that the CpACBP1 gene is mainly expressed during the intracellular developmental stages and that the level increases during parasite development. Immunofluorescence microscopy showed that CpACBP1 is associated with the parasitophorous vacuole membrane (PVM), which implies that this protein may be involved in lipid remodelling in the PVM, or in the transport of fatty acids across the membrane. We also identified two distinct oxysterol binding protein (OSBP)-related proteins (ORPs) from this parasite (CpORP1 and CpORP2). The short-type CpOPR1 contains only a ligand binding (LB) domain, while the long-type CpORP2 contains Pleckstrin homology (PH) and LB domains. Lipid-protein overlay assays using recombinant proteins revealed that CpORP1 and CpORP2 could specifically bind to phosphatidic acid (PA), various phosphatidylinositol phosphates (PIPs), and sulfatide, but not to other types of lipids with simple heads. Cholesterol was not a ligand for these two proteins. CpOPR1 was found mainly on the parasitophorous vacuole membrane (PVM), suggesting that CpORP1 is probably involved in the lipid transport across this unique membrane barrier between parasites and host intestinal lumen. Although Cryptosporidium has two ORPs, other apicomplexans, including Plasmodium, Toxoplasma, and Eimeria, possess only a single long-type ORP, suggesting that this family of proteins may play different roles among apicomplexans.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 6 uppsatser.

Thesis (M.S.)--University of Toledo, 2007 / "In partial fulfillment of the requirements for the degree of Master of Science in Biomedical Sciences." Title from title page of PDF document. Bibliography: p. 37-48, 62-84.

Thesis (Ph. D.)--University of Washington, 1998. / Vita. Includes bibliographical references (leaves [47]-52).

Thesis (doctoral)--Lund University, 1994. / Added t.p. with thesis statement inserted.

Thesis (Ph. D.)--University of California, San Francisco, 2004. / Includes bibliographical references (leaves 101-114). Also available online.

Thesis (doctoral)--Lund University, 1994. / Added t.p. with thesis statement inserted.

Thesis (M.S.)--West Virginia University, 2002. / Title from document title page. Document formatted into pages; contains viii, 60 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 51-55).