Analysis of the catalytic mechanism of juvenile hormone esterase by site-directed mutagenesis

1. 1. Juvenile hormone esterase (JHE) is a serine hydrolase selective for hydrolysis of the conjugated methyl esters of insect juvenile hormones. 2. 2. We have investigated the mechanism of catalytic action of this enzyme by site-directed mutagenesis of the cloned enzyme and expression of the mutants in a baculovirus system. 3. 3. A series of individual mutations of JHE were made to residues possibly involved in catalysis of juvenile hormones, and which are highly conserved m both esterases and lipases. 4. 4. Mutation of the serine residue at position 201to glycine (S201G), or aspartate 173 to asparagine (D173N), or histidine 446 to lysine (H446K), removed all detectable activity and these mutagenized enzymes were determined to be at least 10⁶-fold less active than wild type JHE. 5. 5. Mutation of arginine 47 to histicline (R47H) decreased but did not abolish activity, with K_m essentially unchanged at 66 nM for R47H compared to 34 nM for wild type JHE. 6. 6. The k_cat for R47H was decreased from 103min^-1 for wild type JHE to 1.9 min^-1. 7. 7. In addition, glutamate residue 332 was altered to glutamine (E332Q) and expressed in an Escherichia coli system. 8. 8. This mutation was also found to remove all detectable activity. 9. 9. From the results presented in this study and by comparison of JHE to other serine esterases and Upases, we predict that JHE possesses a Ser₂₀₁-His₄₄₆-Glu₃₃₂ catalytic triad. 10. 10. In addition, aspartate 173 and arginine 47 are essential for the efficient functioning of JHE.