Purification and properties of 1-(hydroxycinnamoyl)-glucose:1-(hydroxycinnamoyl)-glucose hydroxycinnamoyltransferase from radish seedlings

Abstract

A hydroxycinnamoyltransferase (EC 2.3.1.—) which catalyses in vivo the formation of 1,2-di- O-sinapoyl-β- D-glucose has been purified 240-fold from cotyledons of 5-day-old dark-grown seedlings of radish Raphanus sativus L. var. sativus cv. Saxa). The enzyme catalysing a reaction between two identical substrate molecules used 1- O-(hydroxycinnamoyl)-β- D-glucose both as acyl donor and acceptor molecule and exhibited a strict specificity of transfer to the C-2 hydroxyl group of the acceptor. It had an apparent M r , of 55 000 and showed greatest activity at pH 8.0 (50% at pH 7.0 and 9.0) and 42°. Apparent energy of activation was found to be 62 kJ/mol. There was no requirement for divalent cations or thiols. The Michaelis curve did not show a rectangular hyperbola but was slightly sigmoid. A linear curve was obtained on replotting the Michaelis curve according to Hanes, using squared values of substrate concentrations. The S 0.5 for 1-sinapoyl-, 1-feruloyl- and 1-( p-coumaroyl)-glucose were 0.42, 0.40 and 0.58 mM, respectively, and the ratios of the V max/ S 0.5 values were 100:92:45.