Study of regulatory mechanisms of glycolytic and gluconeogenic enzymes
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Many diseases correlate with abnormal glucose metabolism in cells and organisms. For instance, the human M2 isoform of the glycolytic enzyme pyruvate kinase (M2PYK) plays an important role in metabolic reprogramming of tumour cells whereby aerobic glycolysis or the ‘Warburg effect’ supports cell proliferation by accumulating necessary biomass. By contrast, gluconeogenesis may play an important role, as observed in certain types of trypanosomatid parasites (e.g. the amastigote form of Leishmania major) where anabolism is essential for infectious properties. Hence, these glucose metabolising enzymes are important potential drug targets for cancer and trypanosomiasis. However, many aspects of their regulatory mechanisms are still poorly understood. This thesis describes biochemical and structural studies on M2PYK and on L. major fructose-1,6-bisphosphatase (LmFBPase), providing insights into allosteric mechanisms and structure-based drug design for both enzymes. Human PYKs and LmFBPase were expressed and purified from Escherichia coli, and their kinetics were fully characterised. It was shown that certain amino acids regulate the activity of M2PYK allosterically, but in opposite ways, with some being inhibitors and others activators. X-ray crystallographic structures and biophysical data of M2PYK complexes with alanine, phenylalanine, serine or tryptophan reveal an R-/T-state oscillating model of M2PYK involving a 11° rotation of each subunit. In addition, M2PYK was demonstrated to be a redox-sensitive enzyme. Reducing reagents were shown to help maintain the tetramer and prevent its dissociation, and thereby to activate M2PYK, whereas oxidation and nitrosylation reagents functioned in the opposite sense. Nitrosylation assays showed that the main nitrosylated residue is Cys326 of M2PYK, which is located on the tetramer interface. Dynamics and modulator effects of PYKs were further studied by hydrogen–deuterium exchange by mass spectrometry. These observations highlight the important effects of amino acids on M2PYK regulation. M1PYK by contrast, was demonstrated to be a constitutively fully active pyruvate kinase, with minor effects from modulators. The gluconeogenic enzyme fructose-1,6-bisphosphatase (FBPase) is a potential drug target against leishmaniasis. Here we present biochemical and structural studies for LmFBPase, by characterising its activity in a metal-dependent reaction, as well as its inhibition by AMP. The crystal structure of LmFBPase is a homotetramer, composed of monomers with alternating α/β/α/β/α ‘club sandwich’ topologies. In comparison with previously revealed LmFBPase structures, the AMP-complexed structure shows a rotated form of the tetramer. Comparisons of the structures reveal an ‘unlock-androtate’ allosteric mechanism in which AMP binding causes a series of structural changes culminating in an incomplete and non-productive active site. The structure of the effector site of LmFBPase shows a different conformation from human FBPases, thereby offering a potential specific target for Leishmania.