Purification and properties of S-adenosyl- l-methionine:Nicotinic acid- N-methyltransferase from cell suspension cultures of Glycine max L.

Abstract

A soluble enzyme which catalyzes the transfer of the methyl group from S-adenosyl- l-methionine to the nitrogen atom of pyridine-3-car☐ylic acid (nicotinic acid) could be detected in protein preparations from heterotrophic cell suspension cultures of soybean ( Glycine max L.). Enzyme activity was enriched nearly 100-fold by ammonium sulfate precipitation, gel filtration, and ion-exchange chromatography to study kinetic properties. S-Adenosyl- l-methionine:nicotinic acid- N-methyltransferase (EC 2.1.1.7) showed a pH optimum at pH 8.0 and a temperature optimum between 35 and 40 °C. The apparent K M values were determined to be 78 μ m for nicotinic acid and 55 μ m for the cosubstrate. S-Adenosyl- l-homocysteine was a competitive inhibitor of the methyl-transferase with a K I value of 95 μ m. The native enzyme had a molecular mass of about 90 kDa. The catalytic activity was inhibited by reagents blocking SH groups, whereas other divalent cations did not significantly influence the enzyme reaction. The purified methyltransferase revealed a remarkable specificity for nicotinic acid. No other pyridine derivative was a suitable methyl group acceptor. To study a potential methyltransferase activity with nicotinamide as substrate, an additional purification step was necessary to remove nicotinamide amidohydrolase activity from the enzyme preparation. This was achieved by affinity chromatography on S-adenosyl- l-homocysteine-Sepharose thus leading to a 580-fold purified enzyme which showed no methyltransferase activity toward nicotinamide as substrate.