Characterization of acetyl-CoA: Ecdysone 3-acetyltransferase in Schistocerca gregaria larvae

Abstract

Characterization of the acetyltransferase (acetyl-CoA: ecdysone 3-acetyltransferase) which catalyzes the conversion of ecdysone into ecdysone 3-acetate was carried out in gastric caecae of day 7 last instar larvae of Schistocerca gregaria. This enzyme is one of the enzymic systems involved in the inactivation of ecdysteroids. The acetyltransferase exhibited a microsomal subcellular localization, an apparent K_m for ecdysone of 71 μM, a maximal specific activity of 7.2 nmol/min/mg of protein and was inhibited competitively in the presence of 20-hydroxyecdysone with K_i = 68.8 μM. The enzyme required acetyl-CoA as co-substrate for its activity, the apparent K_m for acetyl-CoA being 47.2 μM. Acetic acid could not replace acetyl-CoA as the co-substrate, indicating that the enzyme is an acetyl-CoA: ecdysone acetyltransferase and not a hydrolase. Similarly, esterification of ecdysone was not observed when long-chain fatty acyl-CoA derivatives were substituted as co-substrates. The reaction was linear for 20 min and with protein concentration up to 0.8 mg/ml.

The formation of 20-hydroxyecdysone 3-acetate has been demonstrated in the same microsomal fraction and required also acetyl-CoA as co-substrate. The apparent K_m of the acetyltransferase for 20-hydroxyecdysone was 53.5 μM, revealing that the enzyme had a somewhat stronger affinity for 20-hydroxyecdysone than for ecdysone.