Kemp elimination catalysts by computational enzyme design

  • Robinson P
  • Holbrook K
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The design of new enzymes for reactions not catalysed by naturally occurring biocatalysts is a challenge for protein engineering and is a critical test of our understanding of enzyme catalysis. Here we describe the computational design of eight enzymes that use two different catalytic motifs to catalyse the Kemp elimination-a model reaction for proton transfer from carbon-with measured rate enhancements of up to 10(5) and multiple turnovers. Mutational analysis confirms that catalysis depends on the computationally designed active sites, and a high-resolution crystal structure suggests that the designs have close to atomic accuracy. Application of in vitro evolution to enhance the computational designs produced a >200-fold increase in k(cat)/K(m) (k(cat)/K(m) of 2,600 M(-1)s(-1) and k(cat)/k(uncat) of >10(6)). These results demonstrate the power of combining computational protein design with directed evolution for creating new enzymes, and we anticipate the creation of a wide range of useful new catalysts in the future.

Author-supplied keywords

  • Algorithms
  • Amino Acid Motifs
  • Binding Sites
  • Binding Sites: genetics
  • Catalysis
  • Chemical
  • Computational Biology
  • Computer Simulation
  • Crystallography
  • Directed Molecular Evolution
  • Directed Molecular Evolution: methods
  • Drug Design
  • Drug Evaluation
  • Enzymes
  • Enzymes: chemistry
  • Enzymes: genetics
  • Enzymes: metabolism
  • Kinetics
  • Models
  • Molecular
  • Preclinical
  • Protein Engineering
  • Protein Engineering: methods
  • Quantum Theory
  • Sensitivity and Specificity
  • X-Ray

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  • P. J. Robinson

  • K. A. Holbrook

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