Purification and properties of : Caffeic acid o-methyltransferase from leaves of spinach Beet (Beta vulgaris L.)

1. An enzyme catalysing the methylation of caffeic acid to ferulic acid, using S-adenosyl-l-methionine as methyl donor, has been extracted from leaves of spinach beet and purified 75-fold to obtain a stable prepation. 2. The enzyme showed optimum activity at pH 6.5, and did not require the addition of Mg2+ for maximum activity. 3. It was most active with caffeic acid, but showed some activity with catechol, protocatechuic acid and 3,4-dihydroxybenzaldehyde. The Km for caffeic acid was 68 μM. 4. The Km for S-Adenosyl-l-rmmethionine was 12.5 μM. S-Adenosyl-l-homocysteine (Ki = 4.4 μM) was a competitive inhibitor of S-adenosyl-l-methionine. 5. The synthesis of S-adenosyl-l-homocysteine from adenosine and l-homocysteine and its consequent effect on caffeic acid methylation were demonstrated with a partially-purified preparation from spinach-beet leaves, which possessed both S-adenosyl-l-homocysteine hydrolase (EC 3.3.1.1) and adenosine nucleosidase (EC 3.2.2.7) activities. This preparation was also able to catalyse the rapid breakdown of S-adenosyl-l-homocysteine to adenosine and adenine; the possible significance of this reaction in relieving the inhibition of caffeic acid methylation by S-adenosyl-l-homocysteine is discussed.