High Throughput Glycerate Kinase Activity Assay Using Crude Leaf Extract and Recombinant Enzyme to Determine Kinetic Parameters Km and Vmax Using a Microplate Reader

We describe an assay for measuring the activity of D-glycerate 3-kinase (GLYK) in a 96-well microplate format with the use of a set of coupling enzymes. The assay is appropriate for use with a crude protein extract prepared from leaf tissue and with the recombinant purified enzyme. The 96-well microplate format reduces the needed amounts of reagents and coupling enzymes, making the assay less expensive, high throughput, and suitable for the determination of kinetic parameters Km and Vmax. In addition, we provide a two-step discontinuous assay modified from past work, making it possible to measure the activity of GLYK at temperatures higher than 45 °C.