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Literature summary for 3.1.1.59 extracted from

  • Kamita, S.G.; Wogulis, M.D.; Law, C.S.; Morisseau, C.; Tanaka, H.; Huang, H.; Wilson, D.K.; Hammock, B.D.
    Function of phenylalanine 259 and threonine 314 within the substrate binding pocket of the juvenile hormone esterase of Manduca sexta (2010), Biochemistry, 49, 3733-3742.
    View publication on PubMedView publication on EuropePMC
Search for more literature for 3.1.1.59

Cloned(Commentary)

Cloned (Comment) Organism
recombinant baculovirus AcMsJHE-F259I expressing mutant F259I, recombinant baculovirus AcMsJHE-T314V or recombinant baculovirus pAcUW21-T314V expressing mutant T314V. AcMsJHE-T314V generated by transfection of Sf-21 cells with pAcUW21-T314V and linearized BacPAK6 baculovirus DNA. Wild-type JHE, mutants F259I and T314V produced in insect High Five cells cultured in ESF 921 medium that are inoculated with AcMsJHE7, AcMsJHE-F259I, or AcMsJHE-T314V, respectively Manduca sexta

Protein Variants

Protein Variants Comment Organism
F259I the overall structure of the substrate binding pocket is similar to that of wild-type JHE. Shows a 5.7fold higher kcat for juvenile hormone III as the wild-type. KM of mutant F259I for juvenile hormone III is 3.4fold higher than that of the wild-type. With the saturated form of juvenile hormone III as a substrate, mutant F259I shows a 4.4fold higher kcat and 14fold higher Km in comparison to the wild-type Manduca sexta
T314V no major changes in the overall structure of the substrate binding pocket. The KM of mutant T314V for juvenile hormone III is 2.9fold higher than that of JHE, whereas kcat is 6.4fold higher. Specific activity of mutant T314V for juvenile hormone III diol is not statistically different than that of wild-type for juvenile hormone III Manduca sexta

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.00003
-
 juvenile hormone III wild-type, at 30°C in 50 mM sodium phosphate buffer, pH 7.4, containing 1 mg/mL of bovine serum albumin Manduca sexta
0.000046
-
 saturated juvenile hormone III wild-type, at 30°C in 50 mM sodium phosphate buffer, pH 7.4, containing 1 mg/mL of bovine serum albumin Manduca sexta
0.000086
-
 juvenile hormone III mutant T314V, at 30°C in 50 mM sodium phosphate buffer, pH 7.4, containing 1 mg/mL of bovine serum albumin Manduca sexta
0.000101
-
 juvenile hormone III mutant F259I, at 30°C in 50 mM sodium phosphate buffer, pH 7.4, containing 1 mg/mL of bovine serum albumin Manduca sexta
0.000641
-
 saturated juvenile hormone III mutant F259I, at 30°C in 50 mM sodium phosphate buffer, pH 7.4, containing 1 mg/mL of bovine serum albumin Manduca sexta
0.41
-
 Alpha-naphthyl acetate wild-type, at 30°C in 50 mM sodium phosphate buffer, pH 7.4, containing 1 mg/mL of bovine serum albumin Manduca sexta

Organism

Organism UniProt Comment Textmining
Manduca sexta Q9GPG0
-
-

Purification (Commentary)

Purification (Comment) Organism
expressed wild-type and mutant proteins purified by anion-exchange chromatography. Purification efficiencies of wild-type JHE, mutant F259I, and mutant T314V are 77%, 32%, and 51%, respectively and are purified 4-, 4.4-, and 4.5fold, respectively Manduca sexta

Source Tissue

Source Tissue Comment Organism Textmining

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
500000
-
 purified wild-type, at 30°C in 50 mM sodium phosphate buffer, pH 7.4, containing 0.0005 mM juvenile hormone III and 1 mg/mL of bovine serum albumin Manduca sexta
1050000
-
 purified wild-type, at 30°C in 50 mM sodium phosphate buffer, pH 7.4, containing 0.0005 mM juvenile hormone III diol and 1 mg/mL of bovine serum albumin Manduca sexta
2370000
-
 purified mutant F259I, at 30°C in 50 mM sodium phosphate buffer, pH 7.4, containing 0.0005 mM juvenile hormone III and 1 mg/mL of bovine serum albumin Manduca sexta
4200000
-
 purified mutant T314V, at 30°C in 50 mM sodium phosphate buffer, pH 7.4, containing 0.0005 mM juvenile hormone III and 1 mg/mL of bovine serum albumin Manduca sexta
4880000
-
 purified mutant T314V, at 30°C in 50 mM sodium phosphate buffer, pH 7.4, containing 0.0005 mM juvenile hormone III diol and 1 mg/mL of bovine serum albumin Manduca sexta

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
alpha-naphthyl acetate + H2O
-
 Manduca sexta alpha-naphthol + acetate
-
 ?
juvenile hormone II + H2O
-
 Manduca sexta juvenile hormone II acid + methanol
-
 ?
juvenile hormone III + H2O juvenile hormone III fits very tightly within the substrate binding pocket of JHE. This tight fit places two noncatalytic amino acid residues, Phe-259 and Thr-314, within the appropriate distance and geometry to potentially interact with the R,beta-unsaturated ester and epoxide, respectively, of juvenile hormone III. Neither nucleophilic attack of the resonance-stabilized ester by the catalytic serine nor the availability of a water molecule for attack of the acyl-enzyme intermediate appears to be a rate-determining step in the hydrolysis. Release of the juvenile hormone III acid metabolite from the substrate binding pocket limits the catalytic cycle. Chemical bond strength does not necessarily correlate with how reactive the bond will be to metabolism Manduca sexta juvenile hormone III acid + methanol
-
 ?
juvenile hormone III diol + H2O although hydrogen bonding appears not to occur between juvenile hormone III diol and Thr-314, the hydroxyls of the diol (at C10 and C11) are within hydrogen bonding distance of the main chain carbonyl oxygen atoms of Phe-259 and Phe-311 Manduca sexta ?
-
 ?
saturated juvenile hormone III + H2O alpha,beta-saturated methyl ester Manduca sexta saturated juvenile hormone III acid + methanol
-
 ?

Synonyms

Synonyms Comment Organism
JHE
-
 Manduca sexta
Juvenile hormone esterase
-
 Manduca sexta

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.7
-
 juvenile hormone III wild-type, at 30°C in 50 mM sodium phosphate buffer, pH 7.4, containing 1 mg/mL of bovine serum albumin Manduca sexta
0.7
-
 saturated juvenile hormone III wild-type, at 30°C in 50 mM sodium phosphate buffer, pH 7.4, containing 1 mg/mL of bovine serum albumin Manduca sexta
3.1
-
 saturated juvenile hormone III mutant F259I, at 30°C in 50 mM sodium phosphate buffer, pH 7.4, containing 1 mg/mL of bovine serum albumin Manduca sexta
4
-
 juvenile hormone III mutant F259I, at 30°C in 50 mM sodium phosphate buffer, pH 7.4, containing 1 mg/mL of bovine serum albumin Manduca sexta
4.5
-
 juvenile hormone III mutant T314V, at 30°C in 50 mM sodium phosphate buffer, pH 7.4, containing 1 mg/mL of bovine serum albumin Manduca sexta
22
-
 Alpha-naphthyl acetate wild-type, at 30°C in 50 mM sodium phosphate buffer, pH 7.4, containing 1 mg/mL of bovine serum albumin Manduca sexta

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
53.65
-
 Alpha-naphthyl acetate wild-type, at 30°C in 50 mM sodium phosphate buffer, pH 7.4, containing 1 mg/mL of bovine serum albumin Manduca sexta
4800
-
 saturated juvenile hormone III mutant F259I, at 30°C in 50 mM sodium phosphate buffer, pH 7.4, containing 1 mg/mL of bovine serum albumin Manduca sexta
14000
-
 saturated juvenile hormone III wild-type, at 30°C in 50 mM sodium phosphate buffer, pH 7.4, containing 1 mg/mL of bovine serum albumin Manduca sexta
22000
-
 juvenile hormone III wild-type, at 30°C in 50 mM sodium phosphate buffer, pH 7.4, containing 1 mg/mL of bovine serum albumin Manduca sexta
40000
-
 juvenile hormone III mutant F259I, at 30°C in 50 mM sodium phosphate buffer, pH 7.4, containing 1 mg/mL of bovine serum albumin Manduca sexta
53000
-
 juvenile hormone III mutant T314V, at 30°C in 50 mM sodium phosphate buffer, pH 7.4, containing 1 mg/mL of bovine serum albumin Manduca sexta