Switching Catalysis from Hydrolysis to Perhydrolysis in Pseudomonas fluorescens Esterase

Many serine hydrolases catalyze perhydrolysis, file reversible formation of peracids from carboxylic acids and hydrogen peroxide. Recently. we showed that it Single amino acid Substitution In file alcohol binding pocket, L29P, in Pseudomonas fluorescens (SIK WI) aryl esterase (PFE) increased the specificity constant of PFE for peracetic acid formation > 100-fold [Bernhardt et al. (2005) Angew. Chem., Int. Ed. 44, 2742]. Ill this paper, we extend tills work to address file three following questions. First, what is the molecular basis of the increase in perhydrolysis activity? We previously proposed that the L29P substitution creates it hydrogen bond between the enzyme and hydrogen pet-oxide ill the transition state. Here we report two X-ray Structures of L29P PFE that support this proposal. Both structures show it main chain carbonyl oxygen closer to the active site serine its expected. One structure further shows acetate in the active site ill all orientation consistent with reaction by an acyl-enzyme mechanism. We also detected,In acyl-enzyme intermediate in the hydrolysis of epsilon-caprolactone by mass spectrometry. Second, call we further increase perhydrolysis activity? We discovered that the reverse reaction, hydrolysis of peracetic acid to acetic acid and hydrogen Peroxide, Occurs it( nearly file diffusion limited rate. Since the reverse reaction cannot increase further, neither call the forward reaction. Consistent with this prediction, two variants with additional amino acid Substitutions showed 2-fold higher k(cat), but K-m also increased so the specificity constant, k(cat)/K-m, remained similar. Third, how does the L29P Substitution change the esterase activity? Ester hydrolysis decreased for most esters (75-fold for ethyl acetate) but not for methyl esters. In contrast, L29P PFE catalyzed hydrolysis of epsilon-caprolactone five times more efficiently than wild-type PFE. Molecular modeling Suggests that moving the carbonyl group closer to the active site blocks access for larger alcohol moieties but binds E-caprolactone more tightly. These results are Consistent with file natural function of perhydrolases being, either hydrolysis of peroxycarboxylic acids or hydrolysis of lactones.