Analysis of the catalytic mechanism of juvenile hormone esterase by site-directed mutagenesis.
1. Juvenile hormone esterase (JHE) is a serine hydrolase selective for hydrolysis of the conjugated methyl esters of insect juvenile hormones. 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. A series of individual mutations of JHE were made to residues possibly involved in catalysis of juvenile hormones, and which are highly conserved in both esterases and lipases. 4. Mutation of the serine residue at position 201 to 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 10(6)-fold less active than wild type JHE. 5. Mutation of arginine 47 to histidine (R47H) decreased but did not abolish activity, with Km essentially unchanged at 66 nM for R47H compared to 34 nM for wild type JHE. 6. The kcat for R47H was decreased from 103 min-1 for wild type JHE to 1.9 min-1. 7. In addition, glutamate residue 332 was altered to glutamine (E332Q) and expressed in an Escherichia coli system. 8. This mutation was also found to remove all detectable activity. 9. From the results presented in this study and by comparison of JHE to other serine esterases and lipases, we predict that JHE possesses a Ser201-His446-Glu332 catalytic triad. 10. In addition, aspartate 173 and arginine 47 are essential for the efficient functioning of JHE.
Ward
V K
VK
Department of Entomology, University of California, Davis 95616.
Bonning
B C
BC
Huang
T
T
Shiotsuki
T
T
Griffeth
V N
VN
Hammock
B D
BD
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002265:Carboxylic Ester Hydrolases / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004795:Enzyme Stability; D004950:Esterases / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017386:Sequence Homology, Amino Acid
D004247:DNA; D004950:Esterases; D002265:Carboxylic Ester Hydrolases; C038003:juvenile hormone esterase