Purification and kinetic characterisation 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 selective precipitations, isoelectric focussing, anion exchange and gel filtration chromatography. The KM and Vmax of the purified enzyme for juvenile hormone III (JHIII) hydrolysis are 89 nM and at least 590 nmol/min/mg, respectively. JHE also hydrolyses the artificial substrate alpha-naphthyl acetate with a KM of 120 micro M and a Vmax of at least 70 mumol/min/mg. Competition of JHIII hydrolysis by five juvenile hormones and twenty-four JH analogues showed JHE is highly selective for JHIII and JHIII bisepoxide (JHP3), and both may be in vivo substrates. Binding in the active site of JHE is promoted by structural features found in JHIII and JHB3 including the epoxide groups in their natural orientations, methyl (rather than ethyl) side-chains, and the 2E, 3 double bond that is conjugated with the ester group. Binding is reduced by almost any departure from these structural features of JH. Co-incubation of the haemolymph JH binding protein, lipophorin, with JHE indicates lipophorin might modulate JH hydrolysis by competition for binding of JH.
CSIRO Division of Entomology, Canberra, ACT, Australia. peterca@ento.csiro.au
P M
PM
Campbell
J G
JG
Oakeshott
M J
MJ
Healy
D000438:Alcohols; D000818:Animals; D002265:Carboxylic Ester Hydrolases / Q000302:isolation & purification / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism; D004331:Drosophila melanogaster / Q000201:enzymology*; D004795:Enzyme Stability; D004852:Epoxy Compounds; D005231:Fatty Acids, Unsaturated; D006868:Hydrolysis; D007700:Kinetics; D008074:Lipoproteins / Q000378:metabolism; D008970:Molecular Weight; D009284:Naphthols; D012717:Sesquiterpenes
D000438:Alcohols; D002352:Carrier Proteins; D004852:Epoxy Compounds; D005231:Fatty Acids, Unsaturated; D008074:Lipoproteins; D009284:Naphthols; D012717:Sesquiterpenes; C032830:lipophorin; C032429:methyl farnesoate; C027876:alpha-naphthyl acetate; C036585:juvenile hormone III; D002265:Carboxylic Ester Hydrolases; C038003:juvenile hormone esterase