Abstract
Choline Acetyltransferase (ChAT) synthesizes acetylcholine, the neurotransmitter that carries signals across the neuromuscular junction. Point mutations in ChAT cause motor disorders, apparently by disrupting its active site structure, which is unstable due to many cavities in the enzyme core. One of the large cavities in ChAT was targeted for study and was “filled” by mutating a nearby residue. ChAT with a specific congenital mutant alone as well as ChAT with this congenital mutant plus the cavity‐filling mutation were constructed. The mutants were expressed and purified, thermal stability measured through a thermal shift assay, and activity measurements made using a coupled enzyme activity assay. It was discovered that the induced point mutation did rescue both the thermal stability and overall activity of the enzyme. Also, both the thermal stability assay and activity assay showed that the cavity‐filling mutation surpassed the thermal stability and activity of the wild type enzyme. It was concluded that this cavity is a good target for studying potential ligand binding in hopes of developing therapeutics for the disease caused by certain ChAT mutations.
Published Version
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