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.

Many serine hydrolases catalyze perhydrolysis -the reversible formation of per-acids 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. Angew. Chem. Intl. Ed. 2005, 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 activesite 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 ε-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 two 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 ε-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 ε-caprolactone more tightly. These results are consistent with the natural function of perhydrolases being either hydrolysis of peroxycarboxylic acids or hydrolysis of lactones.* To whom correspondence should be addressed: Romas Kazlauskas: rjk@umn.edu. Fax: +1-612-625-5780. Phone: +1-612-624-5904. Joseph Schrag: joe@bri.nrc.ca. Fax: +1-514-496-5143. Phone: +1-514-496-2557. X-ray coordinates have been deposited in the Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (accession nos. 3hea and 3hi4) Supporting Information Available. The supporting information contains 1) the electrospray ionization mass spectrometry data for detection of acyl-enzyme intermediate, 2) the pH-rate profile of perhydrolysis catalyzed by mutants and wild-type PFE and 3) a discussion of previous experiments to distinguish acyl-enzyme versus a noncovalent mechanisms for PFE This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public Access Author ManuscriptBiochemistry. ...

Derivation of the equation for an equilibrium ratio of products in a dynamic combinatorial library.

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.

Enzyme protection: An irreversible solid‐phase, aqueous peptide coupling resulted in the formation of a library of eight dipeptides, while an irreversible protease‐catalyzed hydrolysis destroyed them. Those dipeptides that bound to carbonic anhydrase were protected from destructions. Six cycles of active ester addition produced only the best‐binding dipeptide (>100:1) in 29 % yield.