title: Partial Purification and Characterization of Hydroxycinnamoyl-Coenzyme A:Tyramine Hydroxycinnamoyltransferase from Cell Suspension Cultures of Solanum tuberosum.
abstract: A pathogen elicitor-inducible soluble acyltransferase (tyramine hydroxycinnamoyltransferase [THT], EC 2.3.1), which catalyzes the transfer of hydroxycinnamic acids from hydroxycinnamoyl-coenzyme A (CoA) esters to tyramine in the formation of N-hydroxycinnamoyltyramine, was partially purified with a 380-fold enrichment and a 6% recovery from cell-suspension cultures of potato (Solanum tuberosum L. cv Datura). The enzyme showed specific activities of 33 mkat (kg protein)-1 (formation of feruloyltyramine). The apparent native Mr was found to be approximately 49,000. Highest activity was at pH 6.8 in K-phosphate. The isoelectric point of the enzyme was approximately pH5.2. The apparent energy of activation was calculated to be 96 kJ mol-1. The enzyme activity was stimulated more than 5-fold by 10 mM Ca2+ or Mg2+. The apparent Km values were 36 [mu]M for feruloyl-CoA and 85 and 140 [mu]M for cinnamoyl- and 4-coumaroyl-CoA, respectively. The Km value for tyramine in the presence of feruloyl-CoA was 22 [mu]M. In the presence of 4-coumaroyl-CoA, however, the Km for tyramine increased to about 230 [mu]M. The mode of action was an iso-ordered bi bi mechanism in which A, B, P, and Q equal hydroxycinnamoyl-CoA, tyramine, N-hydroxycinnamoyltyramine, and CoA, respectively. Thus, the reaction occurred in a ternary complex of the enzyme and substrates. The equilibrium constant of the reaction was determined to be 1.3 x 104. This gave a [delta]G[deg][prime] eq value of -23.5 kJ mol-1.
authors.affiliation: Institut fur Pflanzenbiochemie, Abteilung Sekundarstoffwechsel, Weinberg 3, D-06120, Halle (Saale), Germany(H.H., W.S., D.Strack).
authors.forename: H.
authors.initials: H
authors.lastname: Hohlfeld
authors.forename: W.
authors.initials: W
authors.lastname: Schurmann
authors.forename: D.
authors.initials: D
authors.lastname: Scheel
authors.forename: D.
authors.initials: D
authors.lastname: Strack