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.
| Identifer | oai:union.ndltd.org:USASK/oai:usask.ca:etd-08222007-213001 |
| Date | 24 August 2007 |
| Creators | Cotelesage, Julien Joseph Hubert |
| Contributors | Sanders, David A. R., Pato, Mary D., Khandelwal, Ramji L., Goldie, Hughes, Delbaere, Louis T. J., Warrington, Rob C. |
| Publisher | University of Saskatchewan |
| Source Sets | University of Saskatchewan Library |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://library.usask.ca/theses/available/etd-08222007-213001/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University of Saskatchewan or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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