Here we present the structural, kinetic and biophysical characterization of the novel pyruvate kinase (PaPK) from the hyperthermophile Pyrobaculum aerophilum. The novel catalytic, structural and regulatory properties of this unusual enzyme were investigated, including a potassium-independent catalytic cycle, cooperative binding of ADP, and an apparent absence of any response to known allosteric effectors of conventional pyruvate kinases. This dissertation represents the first published comparison of the structures of pyruvate kinase, in both the apo and ligand bound states, from the same species. This analysis revealed surprisingly few structural differences between the two forms of PaPK, suggesting that the structural basis of cooperativity in pyruvate kinase is more subtle than previously thought. Analysis of the apo structure revealed that PaPK contains a phosphate-binding site in the C domain of PaPK, which appears homologous to the binding site of the 6' phosphate of fructose-1,6-bisphosphate, an allosteric activator of conventional pyruvate kinases. This binding site was probed using an array of biophysical and biochemical techniques. These data suggest that this binding site is a functionally neutral feature of PaPK. Additionally, no significant differences were observed in the active site of PaPK that might account for the unusual potassium-independent catalysis exhibited by PaPK. During routine kinetic analysis of PaPK, the cooperative response of the enzyme was found to be temperature-dependent. These data support a role for dynamics in the cooperative regulation of PaPK.
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