Indole‐3‐Pyruvic Acid as an Intermediate in the Conversion of Tryptophan to Indole‐3‐Acetic Acid. I. Some Characteristics of Tryptophan Transaminase from Mung Bean Seedlings

Seedlings of mung bean (Phaseolus aureus) contain a soluble enzyme capable of converting l‐tryptophan to indole‐3‐pyruvic acid by transamination. The concentration of the enzyme is highest in the stem meristem and primary leaves and lowest in the roots. The enzyme was purified 28.6 fold by ammonium sulphate precipitation, Sephadex G‐200 filtration, and electrophoresis. The isoelectric point of the enzyme protein was pH 6.6. The optimum pH and temperature for the catalytic conversion were ca. 8.5 and 53°C respectively. Using l‐tryptophan and α‐ketoglutarate as substrates Km was found to be 3.3 × 10−4 M and the activation energy 18,270 cal per mole. The enzyme converted only the l‐form of tryptophan, phenylalanine, tyrosine, and histidine. Out of 13 other l‐amino acids tested 8 could be transaminated. Eight α‐keto acids tested could all be used as substrates. High efficiency of an α‐keto acid as an amino group acceptor agreed usually with high efficiency of the corresponding amino acid as a donor. The pari ß‐methyl‐α‐ketoisovaleric acid and isoleucine was an exception to that rule. Addition of pyridoxalphosphate to the reaction mixture was not needed. The indole‐3‐pyruvic acid formed in the reaction was trapped and partly stabilized as its borate complex and measured spectrophotometrically at 327 nm. The keto acid formed was further identified by chromatography of its 2,4‐dinitrophenylhydrazone in 4 solvent systems. When using α‐keto‐glutaric acid as a substrate, the glutamic acid produced was determined by the glutamate dehydrogenase method. The sensitivity of the assay permits enzyme determinations in extracts from 5 mg leaves or 100 mg roots.