PURIFICATION AND KINETIC CHARACTERIZATION OF JUVENILE-HORMONE ESTERASE FROM DROSOPHILA-MELANOGASTER

Juvenile hormone esterase (JHE) from the prepupal stage of Drosophila melanogaster was purified about 429-fold to near homogeneity by select ive precipitations, isoelectric focussing, anion exchange and gel filt ration chromatography. The K-M and V-max of the purified enzyme for ju venile hormone III (JHIII) hydrolysis are 89 nM and at least 590 nmol/ min/mg, respectively. JHE also hydrolyses the artificial substrate alp ha-naphthyl acetate with a K-M of 120 mu M and a V-max of at least 70 mu mol/min/mg. Competition of JHIII hydrolysis by five juvenile hormon es and twenty-four JH analogues showed JHE is highly selective for JHI II and JHIII bisepoxide (JHB(3)), and both may be in vivo substrates. Binding in the active site of JHE is promoted by structural features f ound in JHIII and JHB(3) including the epoxide groups in their natural orientations, methyl (rather than ethyl) side-chains, and the 2E,3 do uble bond that is conjugated with the ester group. Binding is reduced by almost any departure from these structural features of JH. Go-incub ation of the haemolymph JH binding protein, lipophorin, with JHE indic ates lipophorin might modulate JH hydrolysis by competition for bindin g of JH. (C) 1998 Elsevier Science Ltd. All rights reserved.