Hexokinases (HKs), are a group of isoenzymes that are important regulators of human glucose metabolism.  HKs are implicated in a variety of disease states, including diabetes and cancer. Despite the wealth of structural and biochemical data, little is known about the interaction between these isozymes and their ligands. In the present work, we will use predictions from computational models to identify amino acid residues that contribute strongly to ligand binding, and introduce mutations at these locations using site-directed mutagenesis. We will begin the mutational studies using yeast HKI. Yeast HKI has a very similar sequence to human HKIV, and will be used as our model system. Computational models, and previous literature, suggest that aspartate211 is important for binding, therefore the first mutation will be from aspartate to alanine (D211A). Mutations of other active site residues will also be performed to probe the role other residues play in ligand binding. We will then compare the mutated HKI forms with the wildtype HKI to determine if the mutations correspond to different kinetic properties, and different affinities for substrate. We will utilize UV-Vis assays and isothermal  titration calorimetry to find the Michaelis constants (KM) and the association constants (Ka) for the wildtype and mutated forms. Our research will provide a better understanding of the active site interactions important in HKI, as well as the effect of mutations on key residues.

Mutational studies of yeast HKI

247th National Meeting (2014)

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