A liquid chromatography-tandem mass spectrometry-based assay for indole-3-acetic acid-amido synthetase.

Indole-3-acetic acid (IAA) amide conjugates play an important role in balancing levels of free IAA in plant cells. The GH3 family of proteins conjugates free IAA with various amino acids. For example, auxin levels modulate expression of the Oryza sativa (rice) GH3-8 protein, which acts to prevent IAA accumulation by coupling the hormone to aspartate. To examine the kinetic properties of the enzyme, we developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay system. Bacterially expressed OsGH3-8 was purified to homogeneity and used to establish the assay system. Monitoring of the reaction confirms the reaction product as IAA-Asp and demonstrates that production of the conjugate increases proportionally with both time and enzyme amount. Steady-state kinetic analysis using the LC-MS/MS-based assay yields the following parameters: V/E(t)(IAA)=20.3 min(-1), K(m)(IAA)=123 microM, V/E(t)(ATP)=14.1 min(-1), K(m)(ATP)=50 microM, V/E(t)(Asp)=28.8 min(-1), K(m)(Asp)=1580 microM. This is the first assignment of kinetic values for any IAA-amido synthetase from plants. Compared with previously described LC- and thin-layer chromatography (TLC)-based assays, this LC-MS/MS method provides a robust and sensitive means for performing direct kinetic studies on a range of IAA-conjugating enzymes.