Function of phenylalanine 259 and threonine 314 within the substrate binding pocket of the juvenile hormone esterase of manduca sexta

Shizuo G. Kamita, Mark D. Wogulis, Christopher S. Law, Christophe Morisseau, Hiromasa Tanaka, Huazhang Huang, David K. Wilson, Bruce D. Hammock

Research output: Contribution to journalArticle

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Abstract

Juvenile hormone (JH) is a key insect developmental hormone that is found at low nanomolar levels in larval insects. The methyl ester of JH is hydrolyzed in many insects by an esterase that shows high specificity for JH. We have previously determined a crystal structure of the JH esterase (JHE) of the tobacco hornworm Manduca sexta (MsJHE) [Wogulis, M., Wheelock, C. E., Kamita, S. G., Hinton, A. C., Whetstone, P. A., Hammock, B. D., and Wilson, D. K. (2006) Biochemistry 45, 4045-4057]. Our molecular modeling indicates that JH fits very tightly within the substrate binding pocket of MsJHE. 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 α,β- unsaturated ester and epoxide, respectively, of JH. These residues are highly conserved in numerous biologically active JHEs. Kinetic analyses of mutants of Phe-259 or Thr-314 indicate that these residues contribute to the low K M that MsJHE shows for JH. This low KM, however, comes at the cost of reduced substrate turnover. 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 of JH by MsJHE. We hypothesize that the release of the JH acid metabolite from the substrate binding pocket limits the catalytic cycle. Our findings also demonstrate that chemical bond strength does not necessarily correlate with how reactive the bond will be to metabolism.

Original languageEnglish (US)
Pages (from-to)3733-3742
Number of pages10
JournalBiochemistry
Volume49
Issue number17
DOIs
StatePublished - May 4 2010

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Manduca
Juvenile Hormones
Threonine
Phenylalanine
Substrates
Esters
Insects
Insect Hormones
Biochemistry
Molecular modeling
Tobacco
juvenile hormone esterase
Chemical bonds
Epoxy Compounds
Esterases
Metabolites
Metabolism
Serine
Hydrolysis
Crystal structure

ASJC Scopus subject areas

  • Biochemistry

Cite this

Kamita, S. G., Wogulis, M. D., Law, C. S., Morisseau, C., Tanaka, H., Huang, H., ... Hammock, B. D. (2010). Function of phenylalanine 259 and threonine 314 within the substrate binding pocket of the juvenile hormone esterase of manduca sexta. Biochemistry, 49(17), 3733-3742. https://doi.org/10.1021/bi901641x

Function of phenylalanine 259 and threonine 314 within the substrate binding pocket of the juvenile hormone esterase of manduca sexta. / Kamita, Shizuo G.; Wogulis, Mark D.; Law, Christopher S.; Morisseau, Christophe; Tanaka, Hiromasa; Huang, Huazhang; Wilson, David K.; Hammock, Bruce D.

In: Biochemistry, Vol. 49, No. 17, 04.05.2010, p. 3733-3742.

Research output: Contribution to journalArticle

Kamita, SG, Wogulis, MD, Law, CS, Morisseau, C, Tanaka, H, Huang, H, Wilson, DK & Hammock, BD 2010, 'Function of phenylalanine 259 and threonine 314 within the substrate binding pocket of the juvenile hormone esterase of manduca sexta', Biochemistry, vol. 49, no. 17, pp. 3733-3742. https://doi.org/10.1021/bi901641x
Kamita, Shizuo G. ; Wogulis, Mark D. ; Law, Christopher S. ; Morisseau, Christophe ; Tanaka, Hiromasa ; Huang, Huazhang ; Wilson, David K. ; Hammock, Bruce D. / Function of phenylalanine 259 and threonine 314 within the substrate binding pocket of the juvenile hormone esterase of manduca sexta. In: Biochemistry. 2010 ; Vol. 49, No. 17. pp. 3733-3742.
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AU - Kamita, Shizuo G.

AU - Wogulis, Mark D.

AU - Law, Christopher S.

AU - Morisseau, Christophe

AU - Tanaka, Hiromasa

AU - Huang, Huazhang

AU - Wilson, David K.

AU - Hammock, Bruce D.

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