title: Revised molecular basis of the promiscuous carboxylic acid perhydrolase activity in serine hydrolases.
abstract: Several serine hydrolases catalyze a promiscuous reaction: perhydrolysis of carboxylic acids to form peroxycarboxylic acids. The working hypothesis is that perhydrolases are more selective than esterases for hydrogen peroxide over water. In this study, we tested this hypothesis, and focused on L29P-PFE (Pseudomonas fluorescens esterase), which catalyzes perhydrolysis of acetic acid 43-fold faster than wild-type PFE. This hypothesis predicts that L29P-PFE should be approximately 43-fold more selective for hydrogen peroxide than wild-type PFE, but experiments show that L29P-PFE is less selective. The ratio of hydrolysis to perhydrolysis of methyl acetate at different concentrations of hydrogen peroxide fit a kinetic model for nucleophile selectivity. L29P-PFE (β(0)=170  M(-1)) is approximately half as selective for hydrogen peroxide over water than wild-type PFE (β(0)=330  M(-1)), which contradicts the working hypothesis. An alternative hypothesis is that carboxylic acid perhydrolases increase perhydrolysis by forming the acyl-enzyme intermediate faster. Consistent with this hypothesis, the rate of acetyl-enzyme formation, measured by (18)O-water exchange into acetic acid, was 25-fold faster with L29P-PFE than with wild-type PFE, which is similar to the 43-fold faster perhydrolysis with L29P-PFE. Molecular modeling of the first tetrahedral intermediate (T(d)1) suggests that a closer carbonyl group found in perhydrolases accepts a hydrogen bond from the leaving group water. This revised understanding can help design more efficient enzymes for perhydrolysis and shows how subtle changes can create new, unnatural functions in enzymes.
authors.affiliation: University of Minnesota, Department of Biochemistry, Molecular Biology & Biophysics and The Biotechnology Institute, 1479 Gortner Avenue, Saint Paul, MN 55108, USA.
authors.forename: DeLu Tyler
authors.initials: DT
authors.lastname: Yin
authors.forename: Romas J
authors.initials: RJ
authors.lastname: Kazlauskas
mesh_terms: D000085:Acetates / Q000378:metabolism; D002264:Carboxylic Acids / Q000737:chemistry*; D002384:Catalysis; D003198:Computer Simulation; D006861:Hydrogen Peroxide / Q000737:chemistry*; D007700:Kinetics; D008956:Models, Chemical; D015394:Molecular Structure; D011551:Pseudomonas fluorescens / Q000201:enzymology*; D057057:Serine Proteases / Q000737:chemistry / Q000378:metabolism*; D014867:Water / Q000737:chemistry
chemical_list: D000085:Acetates; D002264:Carboxylic Acids; D014867:Water; D006861:Hydrogen Peroxide; D057057:Serine Proteases; C046923:methyl acetate