Development of Surrogate Substrates for Juvenile Hormone Esterase

B. F. Mccutchen, T. Uematsu, A. Szekacs, T. L. Huang, T. Shiotsuki, A. Lucas, B. D. Hammock

Research output: Contribution to journalArticle

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Abstract

Twenty-nine thioester compounds were synthesized to test their effectiveness as surrogate substrates for the insect enzyme, juvenile hormone esterase (JHE). Substrates were designed that resembled the endogenous substrate juvenile hormone (JH), with one common factor being a thioester instead of carboxyl ester found in JH. The principle of the spectrophotometric assay is based on a modification of Ellman′s method. Characterization of the substrates showed that replacement of the carbon atom by a sulfur or oxygen β to the carbonyl of the acyl group of the substrates resulted in an approximate five- to sixfold increase in the rate of hydrolysis by JHE. The specific activities of JHE, porcine liver carboxylesterase, and acetylcholinesterase were determined for the surrogate substrates. While JHE and porcine liver carboxylesterase hydrolyzed several of the substrates, acetylcholinesterase did not produce any detectable hydrolysis of the substrates. Michaelis-Menten kinetic parameters of the surrogate substrates when compared to a previously reported partition assay, utilizing radiolabeled [3H]JH III, indicated that the surrogate substrates have lower affinity as indicated by higher Km values but are more easily hydrolyzed (Vmax) by JHE. Furthermore, optimal reaction conditions for substrate hydrolysis and the spectrophotometric reaction were determined. In addition, first order rate constants for base hydrolysis and critical micelle concentrations were determined for several surrogate substrates. The spectrophotometric assay was also compared with a Vmax and research spectrophotometer, and these two instruments produced almost identical slopes. The relative potency of four transition state inhibitors of JHE was found to be similar with those of the surrogate substrates and the [3H]JH III substrate.

Original languageEnglish (US)
Pages (from-to)231-241
Number of pages11
JournalArchives of Biochemistry and Biophysics
Volume307
Issue number2
DOIs
StatePublished - Dec 1993

Fingerprint

Hydrolysis
Substrates
Juvenile Hormones
Carboxylesterase
Acetylcholinesterase
Swine
Liver
Assays
Micelles
Sulfur
Insects
juvenile hormone esterase
Esters
Carbon
Oxygen
Enzymes
Critical micelle concentration
Research
Spectrophotometers
Kinetic parameters

ASJC Scopus subject areas

  • Molecular Biology
  • Biophysics
  • Biochemistry

Cite this

Mccutchen, B. F., Uematsu, T., Szekacs, A., Huang, T. L., Shiotsuki, T., Lucas, A., & Hammock, B. D. (1993). Development of Surrogate Substrates for Juvenile Hormone Esterase. Archives of Biochemistry and Biophysics, 307(2), 231-241. https://doi.org/10.1006/abbi.1993.1584

Development of Surrogate Substrates for Juvenile Hormone Esterase. / Mccutchen, B. F.; Uematsu, T.; Szekacs, A.; Huang, T. L.; Shiotsuki, T.; Lucas, A.; Hammock, B. D.

In: Archives of Biochemistry and Biophysics, Vol. 307, No. 2, 12.1993, p. 231-241.

Research output: Contribution to journalArticle

Mccutchen, BF, Uematsu, T, Szekacs, A, Huang, TL, Shiotsuki, T, Lucas, A & Hammock, BD 1993, 'Development of Surrogate Substrates for Juvenile Hormone Esterase', Archives of Biochemistry and Biophysics, vol. 307, no. 2, pp. 231-241. https://doi.org/10.1006/abbi.1993.1584
Mccutchen BF, Uematsu T, Szekacs A, Huang TL, Shiotsuki T, Lucas A et al. Development of Surrogate Substrates for Juvenile Hormone Esterase. Archives of Biochemistry and Biophysics. 1993 Dec;307(2):231-241. https://doi.org/10.1006/abbi.1993.1584
Mccutchen, B. F. ; Uematsu, T. ; Szekacs, A. ; Huang, T. L. ; Shiotsuki, T. ; Lucas, A. ; Hammock, B. D. / Development of Surrogate Substrates for Juvenile Hormone Esterase. In: Archives of Biochemistry and Biophysics. 1993 ; Vol. 307, No. 2. pp. 231-241.
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