利用DPI技术比较E.coliKARI野生型和变异型与底物结合效率的差异.pdfVIP

Application Note 009 Comparison of Substrate-Binding Efficiency of Wild Type and Mutant E. coli KARI Introduction Dual Polarisation Interferometry (DPI) offers a truly quantitative analytical technique, rather than a simple ‘mass sensor’ response, providing real time, absolute density measurements and structural dimensions of immobilised proteins at high resolution (1) ® . Farfield’s AnaLight DPI instrument range is an important enabling technology for the rapid and sensitive monitoring of both structural and functional events during protein interactions. Ketol-acid reductoisomerase (KARI) is involved in the biosynthesis of branched-chain amino acids in plants and micro-organisms. It catalyzes two separate reactions at a common active site, an isomerisation and a reduction. KARI binds NADPH and then effects the rearrangement of 2-acetolactate to 3-hydroxy-3-methyl-2-ketobutyrate 2+ (HMKB) as part of the amino acid synthesis pathway. There is an absolute requirement for Mg . The bound HMKB is then reduced to 2,3-dihydroxy-3-methyl butyrate by NADPH. It has been proposed that if NADP+ is substituted for NADPH, the isomerisation reaction can proceed but not the subsequent reduction reaction (2). A mutant form of E.coli KARI, D217E, shows impaired substrate binding and no isomerase, reductase or reductisomerase activity in standard assay (2). + In these experiments, DPI was used to compare the structural consequences of the binding of NADPH, NADP and 2-acetolactate to both wild type and D217E mutant KARI.