Abstract
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The bacterial phosphotriesterases catalyze hydrolysis of the pesticide paraoxon with very fast
turnover rates and are thought to be near to their evolutionary limit for this activity. To test whether the
naturally evolved turnover rate could be improved through the incorporation of unnatural amino acids and
to probe the role of peripheral active site residues in nonchemical steps of the catalytic cycle (substrate
binding and product release), we replaced the naturally occurring tyrosine amino acid at position 309 with
unnatural L-(7-hydroxycoumarin-4-yl)ethylglycine (Hco) and L-(7-methylcoumarin-4-yl)ethylglycine amino
acids, as well as leucine, phenylalanine, and tryptophan. Kinetic analysis suggests that the 7-hydroxyl
group of Hco, particularly in its deprotonated state, contributes to an increase in the rate-limiting product
release step of substrate turnover as a result of its electrostatic repulsion of the negatively charged
4-nitrophenolate product of paraoxon hydrolysis. The 8-11-fold improvement of this already highly efficient
catalyst through a single rationally designed mutation using an unnatural amino acid stands in contrast to
the difficulty in improving this native activity through screening hundreds of thousands of mutants with
natural amino acids. These results demonstrate that designer amino acids provide easy access to new
and valuable sequence and functional space for the engineering and evolution of existing enzyme functions.