Understanding substrate binding and movement in proline catabolic enzymes
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Proline catabolism is the breakdown of proline to glutamate. This is catalyzed by two enzymes that can be either distinct, monofunctional enzymes or fused into one bifunctional enzyme. When the two enzymes are fused together they are linked by a large, internal, water filled cavity that can channel the intermediate between the two active sites. Using crystallography, kinetics, ligand soaking, and small angle X-ray scattering, this work studied substrate binding in the active sites and movement of the intermediate between active sites within the bifunctional enzyme. This work highlighted the importance of key residues involved in substrate binding, oligomerization, product release and even intermediate trafficking. Mutations of key residues highlighted gave a more in-depth look at the path the intermediate takes in the internal chamber, by blocking off different segments of the channel. Proline is soluble at high concentrations and was used to soak crystals of proline catabolic enzymes. High concentration soaks led to movements in the enzymes that were unexpected. These high concentrations were obtainable due to proline's cryoprotective capability. This was further studied on enzymes that are not involved in proline metabolism and it was found that proline can be used, generally, as a cryoprotectant for macromolecular crystallography. The diversity of oligomeric state found in this family of states is addressed here, with plenty of data establishing the rules of higher oligomeric states found in close homologous enzymes.
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This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 License.