Kemp elimination catalysts by computational enzyme design

Nature. 2008 May 8;453(7192):190-5. doi: 10.1038/nature06879. Epub 2008 Mar 19.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

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


  • Enzymes

Associated data

  • PDB/2RKX