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A Single Mutation in a Regulatory Protein Produces Evolvable Allosterically Regulated Catalyst of Nonnatural Reaction


  • We thank Prof. Robert P. Doyle for providing access to CD instrument. The Spectroscopy Support Facility at FCCC provided access to a 600 MHz NMR spectrometer.


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It only takes one mutation: A strategically placed single mutation in a non-enzymatic protein scaffold produced AlleyCat, a small, allosterically regulated catalyst of Kemp elimination. In only seven rounds of directed evolution the enzymatic efficiency of the original 74 amino acid residue catalyst was improved more than 220-fold to achieve a kcat value higher than that of catalytic antibodies for the same reaction, still preserving allosteric regulation.