How to check enzyme activity of Tryptophan aminotransferase?

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(Last Updated On: July 20, 2020)
Tryptophan aminotransferase
Tryptophan aminotransferase enzyme catalyzing the conversion of tryptophan to indole-3-pyruvate.

Tryptophan aminotransferase is an enzyme that catalyzes the transamination of L-tryptophan. It requires pyridoxal phosphate as a cofactor and an amino group acceptor alpha-ketoglutarate. Tryptophan aminotransferase catalyzes the first step of indole-3-acetic acid (an auxin) biosynthesis and its product is indole-3-pyruvate.

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Indole-3-acetic acid is a plant growth hormone that can be synthesized through four major pathways and the indole-3-pyruvate pathway is one of the most common pathways. In the indole-3-pyruvate pathway, tryptophan is first converted to indole-3-pyruvate catalyzed by tryptophan aminotransferase and then indole-3-pyruvate is converted to indole-3-acetaldehyde catalyzed by indole-3-pyruvate decarboxylase.

Here, I have mentioned a simple protocol to check the enzyme activity of tryptophan aminotransferase. First, take 1 ml of the crude enzyme (pH 7) and supplement it with 10 µmol α-ketoglutarate, 0.03 µmol of pyridoxal phosphate. Start the reaction by adding 10 µmol of L-tryptophan in a total reaction mixture of 1.4 ml.

Now, incubate the reaction mixture for 1 hour at 30 ºC and read the absorbance at 305 nm. The formation of indole-3-pyruvic acid can be analyzed based on the formation of the enol form of indole-3-pyruvic acid at neutral pH and the absorbance of 0.17 units can be correlated with the formation of 1nmol/ml of product (Speedie et al., 1975).

Reference

Speedie MK, Hornemann U, Floss HG.1975. Isolation and characterization of tryptophan transaminase and indole pyruvate C-methyltransferase. Enzymes involved in indolmycin biosynthesis in Streptomyces griseus. Journal of Biological Chemistry 250(19):7819-7825.

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