PURIFICATION, CHARACTERIZATION AND PARTIAL AMINO-ACID SEQUENCING OF HYDROXYCINNAMOYL-COA-TYRAMINE N-(HYDROXYCINNAMOYL)TRANSFERASE FROM TOBACCO CELL-SUSPENSION CULTURES

We report the purification of hydroxycinnamoyl-CoA :tyramine N-(hydrox ycinnamoyl)transferase (THT) to apparent homogeneity in 12% yield from tobacco (Nicotiana tabacum L. cv. Xanthi) cell-suspension cultures el icited with a commercial preparation of pronase. The purification proc edure employs only four chromatography steps and takes advantage of th e fact that the transferase binds tightly both to phenyl-Sepharose and to hydroxyapatite. The native enzyme has a pI of 5.2 and consists of two identical or Very similar subunits of approximately 24 kDa. The pu rified enzyme can synthesise a wide range of amides due to its relativ ely low specificity for cinnamoyl-CoA derivatives and hydroxyphenethyl amines, but its best substrates are tyramine and feruloyl-CoA. THT fol lows Michaelis-Menten kinetics in the presence of low concentrations o f feruloyl-CoA but negative cooperativity occurs when this concentrati on increases above 2.5 mu M, resulting in a marked decrease of the aff inity for tyramine. Large deviations from Michaelis-Menten kinetics ar e also observed when 3-methoxytyramine is used as acyl acceptor. The a ctivity of tobacco THT is not affected by the addition of CaCl2 or MgC l2 but its maximal velocity is increased up to twofold by addition of ethanol to the assay mixture. It is inhibited in vitro by L-tyrosine b enzyl ester, which binds reversibly to the tyramine-binding site. Expe riments performed using L-tyrosine benzyl ester and caffeoyl-CoA as in hibitors confirm that feruloyl-CoA is the first substrate to add to th e transferase in an ordered bi-bi mechanism. Part of the amino acid se quence of the transferase, elucidated by microsequencing of tryptic pe ptides, is also described.