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Structural and Functional Studies of Phosphoenolpyruvate Carboxykinase

by Cotelesage, Julien Joseph

Abstract (Summary)
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.

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 Escherichia coli; one was a complex of ATP, Mg2+ and CO2, the second structure was an ATP, Mg2+, Mn2+, CO2 and oxaloacetate complex and, the third E. coli structure was a Lys213Ser mutant complexed with ATP, Mg2+and Mn2+. Two Anaerobiospirillum succiniciproducens PCK crystal structures were also solved; one was in the native form and the other was an ATP-Mg2+-Mn2+-oxalate complex.

In the E. coli-PCK-ATP-Mg2+-CO2 crystal complex structure, the observed location of CO2 was in agreement with a previously determined E. coli PCK-CO2 crystal structure, which incorporated CO2 into the structure by a different technique. The findings from the E. coli PCK-ATP-Mg2+-CO2 crystal structure allowed the reaction mechanism presented in this work to be proposed.

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.

Previous kinetic studies on the E. coli 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.

The two crystal structures of A. succiniciproducens 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 A. succiniciproducens 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.

Bibliographical Information:

Advisor:Warrington, Rob; Sanders, David A. R.; Pato, Mary D.; Khandelwal, Ramji L.; Goldie, Hughes; Delbaere, Louis

School:University of Saskatchewan

School Location:Canada - Saskatchewan

Source Type:Master's Thesis

Keywords:carboxylation carbon dioxide binding co2 domain movement

ISBN:

Date of Publication:08/24/2007

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