Switching catalysis from hydrolysis to perhydrolysis in Pseudomonas fluorescens esterase.

Many serine hydrolases catalyze perhydrolysis, the reversible formation of peracids from carboxylic acids and hydrogen peroxide. Recently, we showed that a single amino acid substitution in the 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]. In this paper, we extend this work to address the three following questions. First, what is the molecular basis of the increase in perhydrolysis activity? We previously proposed that the L29P substitution creates a hydrogen bond between the enzyme and hydrogen peroxide in the transition state. Here we report two X-ray structures of L29P PFE that support this proposal. Both structures show a main chain carbonyl oxygen closer to the active site serine as expected. One structure further shows acetate in the active site in an orientation consistent with reaction by an acyl-enzyme mechanism. We also detected an acyl-enzyme intermediate in the hydrolysis of epsilon-caprolactone by mass spectrometry. Second, can we further increase perhydrolysis activity? We discovered that the reverse reaction, hydrolysis of peracetic acid to acetic acid and hydrogen peroxide, occurs at nearly the diffusion limited rate. Since the reverse reaction cannot increase further, neither can 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 epsilon-caprolactone more tightly. These results are consistent with the natural function of perhydrolases being either hydrolysis of peroxycarboxylic acids or hydrolysis of lactones.

Receptor-assisted combinatorial chemistry: thermodynamics and kinetics in drug discovery.

Current drug discovery using combinatorial chemistry involves synthesis followed by screening, but emerging methods involve receptor-assistance to combine these steps. Adding stoichiometric amounts of receptor during library synthesis alters the kinetics or thermodynamics of the synthesis in a way that identifies the best-binding library members. Three main methods have emerged thus far in receptor-assisted combinatorial chemistry: dynamic combinatorial libraries, receptor-accelerated synthesis, and a new method, pseudo-dynamic libraries. Pseudo-dynamic libraries apply both thermodynamics and kinetics to amplify library members to easily observable levels, and attain selectivity heretofore unseen in receptor-assisted systems.

Structure of an aryl esterase from Pseudomonas fluorescens.

The structure of PFE, an aryl esterase from Pseudomonas fluorescens, has been solved to a resolution of 1.8 A by X-ray diffraction and shows a characteristic alpha/beta-hydrolase fold. In addition to catalyzing the hydrolysis of esters in vitro, PFE also shows low bromoperoxidase activity. PFE shows highest structural similarity, including the active-site environment, to a family of non-heme bacterial haloperoxidases, with an r.m.s. deviation in 271 C(alpha) atoms between PFE and its five closest structural neighbors averaging 0.8 A. PFE has far less similarity (r.m.s. deviation in 218 C(alpha) atoms of 5.0 A) to P. fluorescens carboxyl esterase. PFE favors activated esters with small acyl groups, such as phenyl acetate. The X-ray structure of PFE reveals a significantly occluded active site. In addition, several residues, including Trp28 and Met95, limit the size of the acyl-binding pocket, explaining its preference for small acyl groups.

Amplification of screening sensitivity through selective destruction: theory and screening of a library of carbonic anhydrase inhibitors.

A new method for identifying enzyme inhibitors is to conduct their synthesis in the presence of the targeted enzyme. Good inhibitors form in larger amounts than poorer ones because the binding either speeds up synthesis (target-accelerated synthesis) or shifts the synthesis equilibrium (dynamic combinatorial libraries). Several groups have successfully demonstrated this approach with simple systems, but application to larger libraries is challenging because of the need to accurately measure the amount of each inhibitor. In this report, we dramatically simplify this analysis by adding a reaction that destroys the unbound inhibitors. This works similar to a kinetic resolution, with the best inhibitor being the last one remaining. We demonstrate this method for a static library of several sulfonamide inhibitors of carbonic anhydrase. Four sulfonamide-containing dipeptides, EtOC-Phe(sa)-Phe (4a), EtOC-Phe(sa)-Gly (4b), EtOC-Phe(sa)-Leu (4c) and EtOC-Phe(sa)-Pro (4d), were prepared and their inhibition constants measured. These inhibitors migrated to the carbonic anhydrase compartment of a two-compartment vessel. Although higher concentrations of the better inhibitors were observed in the carbonic anhydrase compartment, the concentration differences were small (1.83:1.71:1.54:1.46:1 for 4a:4b:4c:4d:5, where 5 is a noninhibiting dipeptide EtOC-Phe-Phe). Addition of a protease rapidly cleaved the weaker inhibitors (4d and 5). Intermediate inhibitor 4c was cleaved at a slower rate, and at the end of the reaction, only 4a and 4b remained. In a separate experiment, the ratio of 4a to 4b was found to increase over time to a final ratio of nearly 4:1. This is greater than the ratio of their inhibition constants (approximately 2:1). The theoretical model predicts that these ratios would increase even further as the destruction proceeds. This removal of poorer inhibitors simplifies identification of the best inhibitor in a complex mixture.