Juvenile hormone diol kinase. I. Purification, characterization, and substrate specificity of juvenile hormone-selective diol kinase from Manduca sexta.
Manduca sexta juvenile hormone diol kinase (JHDK) catalyzes the conversion of juvenile hormone (JH) diol to JH diol phosphate. JHDK may be the first example of a phosphotransferase directly involved in the catabolism and inactivation of a lipid-soluble hormone. JHDK is an enzyme crucial for secondary metabolism of JH and possesses high specificity and catalytic efficiency for JH diol. In this study, the purification and characterization of native JHDK are described; its enzymatic properties are examined; and its role in cellular JH metabolism is explored. Using a variety of potential substrates, we show that JHDK has a preference for ATP, but will catalyze the formation of JH diol phosphate with GTP as the phosphate donor. JHDK has a nanomolar K(m) for JH I diol and a low micromolar value for MgATP. JH II and III diols also serve as phosphate acceptors with low micromolar K(m), whereas other diol derivatives of terpenoid esters structurally similar to JH metabolites are not phosphorylated. The reaction proceeds via a sequential Bi Bi mechanism. JHDK is active as a homodimer with a subunit molecular mass of 20 kDa. JHDK binds 5'-p-fluorosulfonylbenzoyladenosine and is inhibited by micromolar levels of Ca2+.
Maxwell
Robert A
RA
Department of Biochemistry, University of Nevada, Reno, Nevada 89557, USA.
Welch
William H
WH
Schooley
David A
DA
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002845:Chromatography; D002851:Chromatography, High Pressure Liquid; D007605:Juvenile Hormones / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D018402:Manduca / Q000201:enzymology*; D008956:Models, Chemical; D010770:Phosphotransferases / Q000737:chemistry; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry* / Q000302:isolation & purification*; D013379:Substrate Specificity
D007605:Juvenile Hormones; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C460370:juvenile hormone diol kinase; D008274:Magnesium; D002118:Calcium