The Characterization of the Potential Structural, Allosteric, and Rheostatic Behavior of a New Pyruvate Kinase Homologue




Vela, Kathryn

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Human liver pyruvate kinase (hlPYK) is an essential protein that serves as the catalyst for the last step in glycolysis, the formation of pyruvate and ATP from phosphoenolpyruvate (PEP) and ADP, and is regulated by fructose 1,6-bisphosphate (FBP). The regulation of this enzyme is important in order to maintain cellular homeostasis to prevent diabetic symptoms, as well as to maintain hormone levels in the body. Zymomonas mobilis pyruvate kinase (ZmPYK), a homologue of hlPYK, has been the subject of study because of a phosphate ion bound in the site analogous to the 6'-phosphate site of FBP in hlPYK. This enzyme is unique in that it is dimeric in solution (via gel filtration chromatography), but crystalizes as a tetramer as seen in all other homologues. This can be explained through the buried surface area of the ZmPYK interfaces being significantly smaller than that of hlPYK, and yet tetramerization provides an accessible low energy crystallization pathway. Circular Dichroism (CD) spectra was used to measure melting temperatures of hlPYK and ZmPYK to identify the changes (or lack thereof) in melting temperature upon binding of FBP or the phosphate ion had. hlPYK showed clear changes in both secondary structure and in melting temperatures upon the binding of FBP. In contrast, ZmPYK showed difference in secondary structure and melting temperatures in the presence and absence of the phosphate ion, revealing its inability to tune its activity by increasing stability of the protein. This allows us to conclude that the phosphate ion plays neither a structural or allosteric role in ZmPYK, but rather adventitiously binds to the protein. We hypothesize that ZmPYK does not have sufficient protein stability to tune its activity as is seen for rheostat positions in the allosteric homologues.


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Allosterism, Circular Dichroism, Crystallography, Enzyme, Pyruvate Kinase, Rheostat