Abstract
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 Km essentially unchanged at 66 nM for R47H compared to 34 nM for wild type JHE. 6. 6. The kcat 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.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 1933-1941 |
| Number of pages | 9 |
| Journal | International Journal of Biochemistry |
| Volume | 24 |
| Issue number | 12 |
| DOIs | |
| State | Published - 1992 |
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- Biochemistry
Cite this
Analysis of the catalytic mechanism of juvenile hormone esterase by site-directed mutagenesis. / Ward, Vernon K.; Bonning, Bryony C.; Huang, Tien; Shiotsuki, Takahiro; Griffeth, Valerie N.; Hammock, Bruce D.
In: International Journal of Biochemistry, Vol. 24, No. 12, 1992, p. 1933-1941.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Analysis of the catalytic mechanism of juvenile hormone esterase by site-directed mutagenesis
AU - Ward, Vernon K.
AU - Bonning, Bryony C.
AU - Huang, Tien
AU - Shiotsuki, Takahiro
AU - Griffeth, Valerie N.
AU - Hammock, Bruce D.
PY - 1992
Y1 - 1992
N2 - 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 Km essentially unchanged at 66 nM for R47H compared to 34 nM for wild type JHE. 6. 6. The kcat 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.
AB - 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 Km essentially unchanged at 66 nM for R47H compared to 34 nM for wild type JHE. 6. 6. The kcat 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.
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UR - http://www.scopus.com/inward/citedby.url?scp=0026486907&partnerID=8YFLogxK
U2 - 10.1016/0020-711X(92)90289-D
DO - 10.1016/0020-711X(92)90289-D
M3 - Article
C2 - 1473606
AN - SCOPUS:0026486907
VL - 24
SP - 1933
EP - 1941
JO - International Journal of Biochemistry and Cell Biology
JF - International Journal of Biochemistry and Cell Biology
SN - 1357-2725
IS - 12
ER -