PARTIAL-PURIFICATION AND CHARACTERIZATION OF HYDROXYCINNAMOYL-COENZYME A-TYRAMINE HYDROXYCINNAMOYLTRANSFERASE FROM CELL-SUSPENSION CULTURES OF SOLANUM-TUBEROSUM

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 M(r) 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 pH 5.2. The apparent energy of activation was calculated to be 96 kj mel(-1). The enzyme activity was stimulated more than 5-fold by 10 mM Ca2+ or Mg2+. The apparent K-m values were 36 mu M for feruloyl-CoA and 85 and 140 mu M for cinnamoyl- and 4-coumaroyl-CoA, respectively. The K-m value for tyramine in the presence of feruloyl-CoA was 22 mu M In the presence of 4-coumaroyl-CoA, however, the K-m 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 10(4). This gave a Delta G(O)'( )(eq) value of - 23.5 kJ mol(-1).