Web Release Date: April 26,
Development of Small Designer Aldolase Enzymes: Catalytic Activity, Folding,
and Substrate Specificity
The Skaggs Institute for Chemical Biology and Departments of Chemistry and Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037
Received February 4, 2005
Revised Manuscript Received March 22, 2005
Abstract:
Small (24-35 amino acid residues) peptides that catalyze carbon-carbon bond transformations
including aldol, retro-aldol, and Michael reactions in aqueous buffer via an enamine mechanism have
been developed. Peptide phage libraries were created by appending six randomized amino acid residues
to the C-terminus or to the N-terminus of an 18-mer 
-helix peptide containing lysine residues. Reaction-based selection with 1,3-diketones was performed to trap the amino groups of reactive lysine residues
that were necessary for the catalysis via an enamine mechanism by formation of stable enaminones. The
selected 24-mer peptides catalyzed the reactions with improved activities. The improved activities were
correlated with improved folded states of the peptides. The catalyst was then improved with respect to
substrate specificity by appending a phage display-derived substrate-binding module. The resulting 35-mer peptide functioned with a significant proportion of the catalytic proficiency of larger protein catalysts.
These results indicate that small designer enzymes with good rate acceleration and excellent substrate
specificity can be created by combination of design and reaction-based selection from libraries.
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