Juvenile hormone epoxide hydrolase. Photoaffinity labeling, purification, and characterization from tobacco hornworm eggs.
Juvenile hormone epoxide hydrolase (JHEH), which may play a pivotal role in regulating insect juvenile hormone (JH) titer along with JH esterase, was identified in tobacco hornworm (Manduca sexta) eggs by using photoaffinity analogs of JHs. The UV light-induced covalent labeling with [3H]epoxyhomofarnesyl diazoacetate, a JHII analog, revealed a membrane-associated 50-kDa protein that was selectively and specifically labeled. This 50-kDa protein was copurified 171-fold with the JHEH activity to homogeneity through DEAE-Sephacel, Mono Q, and hydroxylapatite columns, which led us to conclude that the labeled 50-kDa protein was a JHEH. The steady-state kinetics of the purified microsomal JHEH showed that it followed Michaelis-Menten kinetics with Km values of 0.61, 0.55, and 0.28 microM for JHI, II, and III, respectively, and that JHIII showed a significantly higher Vmax than JHI or JHII. JH acid was also converted to the corresponding diol at a rate 4-fold slower than the corresponding JH. Thus, the differences in the binding of substrate and the rate of turnover by JHEH were affected by the epoxyfarnesoate ester moiety of JH and the difference between the cis-11-methyl group of JHIII versus the cis-11-ethyl group of JHI and II. Purified JHEH showed optimal enzyme activity at pH 7.5-8.5. Interestingly, the presence of recombinant M. sexta JH binding protein (JHBP) dramatically decreased the degradation of JH by JHEH in vitro. Since the cytosolic JHBP in eggs closely resembles the hemolymph JHBP, we suggest that cytosolic JHBP may play a role in protecting JHs from JHEH in vivo. Furthermore, JHEH may play a significant role in the secondary metabolism of JH acid generated by JH esterase.
Touhara
K
K
Department of Chemistry, University at Stony Brook, New York 11794-3400.
Prestwich
G D
GD
D000345:Affinity Labels / Q000378:metabolism; D000818:Animals; D002352:Carrier Proteins / Q000494:pharmacology; D002458:Cell Fractionation; D002845:Chromatography; D002852:Chromatography, Ion Exchange; D003979:Diazonium Compounds / Q000378:metabolism; D017886:Durapatite; D004591:Electrophoresis, Polyacrylamide Gel; D004851:Epoxide Hydrolases / Q000302:isolation & purification / Q000378:metabolism*; D005204:Farnesol / Q000031:analogs & derivatives / Q000378:metabolism; D005260:Female; D006863:Hydrogen-Ion Concentration; D006882:Hydroxyapatites; D019476:Insect Proteins; D007605:Juvenile Hormones / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D009036:Moths / Q000201:enzymology*; D009865:Oocytes / Q000201:enzymology*; D013347:Subcellular Fractions / Q000201:enzymology; D014461:Ultracentrifugation
D000345:Affinity Labels; D002352:Carrier Proteins; D003979:Diazonium Compounds; D006882:Hydroxyapatites; D019476:Insect Proteins; D007605:Juvenile Hormones; C035606:juvenile hormone-binding protein, insect; D005204:Farnesol; D017886:Durapatite; C066480:epoxyhomofarnesyl diazoacetate; D004851:Epoxide Hydrolases; C055698:juvenile hormone epoxide hydrolase