A fluorescent sensor of the phosphorylation state of nucleoside diphosphate kinase and its use to monitor nucleoside diphosphate concentrations in real time

A sensor for purine nucleoside diphosphates in solution based on nucleoside diphosphate kinase (NDPK) has been developed. A single cysteine was introd uced into the protein and labeled with the environmentally sensitive fluoro phore, N-[2-(iodoacetamido)ethyl]-7-diethylaminocoumarin-3-carboxamide. The resultant molecule shows a 4-fold fluorescence increase when phosphorylate d on His117; this phosphorylation is on the normal reaction pathway of the enzyme. The emission maximum of the phosphoenzyme is at 475 nm, with maximu m excitation at 430 nm. The fluorescent phosphorylated NDPK is used to meas ure the amount of ADP and the unphosphorylated to measure ATP. The labeled protein is phosphorylated to >90%, and the resultant molecule is stable on ice or can be stored at -80 degreesC. The fluorescence responds to the frac tion of protein phosphorylated and so to the equilibrium between ADP plus N DPK similar toP and ATP plus NDPK. In effect, the sensor measures the ADP/A TP concentration ratio. The enzyme has a broad specificity for the purine o f the nucleotides, so the sensor also can measure GDP/GTP ratios, The fluor escence and kinetic properties of the labeled protein are described. The bi nding rate constants of nucleotides are similar to 10(5) M-1 s(-1), and the fluorescence change is at > 200 s(-1) when the ADP concentration is >1 mM. Results are presented with two well-defined systems, namely, the kinetics of ADP release from myosin subfragment 1 and GDP release from the small G p rotein, human rho. The results obtained with this novel sensor agree with t hose from alternate methods and demonstrate the applicability for following micromolar changes in nucleoside diphosphate in real time.