Development of surrogate substrates for juvenile hormone esterase.
1993
McCutchen B.F. | Uematsu T. | Szekacs A. | Huang T.L. | Shiotsuki T. | Lucas A. | Hammock B.D.
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 beta 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.
اظهر المزيد [+] اقل [-]الكلمات المفتاحية الخاصة بالمكنز الزراعي (أجروفوك)
المعلومات البيبليوغرافية
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