Indole-3-pyruvate decarboxylase is an important enzyme involved in the biosynthesis of indole-3-acetic acid, a plant growth hormone. The indole-3-acetic acid biosynthetic pathway involving indole-3-pyruvate decarboxylase is called an indole-3-pyruvate pathway and indole-3-pyruvate is the key intermediate of this pathway.
The first enzyme of this pathway is the tryptophan aminotransferase which converts tryptophan to indole-3-pyruvate. Indole-3-pyruvate decarboxylase requires thiamine pyrophosphate and MgCl2 as cofactors to work. Here, I have mentioned the simple protocol for the enzyme activity assay of indole-3-pyruvate decarboxylase. I Hope, this protocol will help you do your research on enzyme activity involving indole-3-pyruvate decarboxylase.
Take 0.1 ml of crude enzyme extract and mix it with 2.9 ml of buffer solution of pH 6.5 containing 10 mM potassium phosphate, 1 mM MgCl2, and 0.1 mM Thiamine pyrophosphate (TPP). Start the reaction by adding 30 µl of 20 mM of IPyA (dissolved in ethanol).
Incubate the reaction mixture at 25 ºC for 10 minutes. After incubation, stop the reaction by adding 6 ml of 0.1 N HCl containing 50 % ethanol. The amount of IAAld and Tryptophol formed and the amount of IPyA consumed can be measured with the HPLC using a C-18 column.
Elute the column with 50 % ethanol in 5 % acetic acid with a flow rate of 0.7 ml per minute and monitor the reading at 280 nm excitation and 350 nm emission wavelengths. For your kind of information, one unit of enzyme activity is defined as the amount of enzyme that catalyzes the production of 1 µml of the total amount of IAAld plus the double amount of tryptophol per minute under the above condition (Koga et al. 1992).
Reference:
Koga J, Adachi T, Hidaka H.1992. Purification and characterization of indole pyruvate decarboxylase. A novel enzyme for indole-3-acetic acid biosynthesis in Enterobacter cloacae. Journal of Biological Chemistry 267(22):15823-15828.
