Determination of enzyme/substrate specificity constants using a multiple substrate ESI-MS assay

摘要: The traditional method used to investigate the reaction specificity of an enzyme with different substrates is perform individual kinetic measurements. In this case, a series varied concentrations are required study each substrate and non-regression analysis program several times obtain all constants for comparison. To avoid large amount experimental materials, long time, redundant data processing procedures involved in method, we have developed novel strategy rapid determination using one system containing multiple competing substrates. multiplex assay electrospray ionization mass spectrometry (ESI-MS) technique was simultaneous quantification products steady-state kinetics model established efficient constant calculation. investigated bacterial sulfotransferase NodH (NodST), which host specific nod gene product that catalyzes sulfate group transfer from 3′-phosphoadenosine 5′-phosphosulfate (PAPS) natural Nod factors or synthetic chitooligosaccharides. Herein, NodST four chitooligosaccharide acceptor chain length (chitobiose, chitotriose, chitotetraose, chitopentaose) determined by both measurements new ESI-MS assay. results obtained two methods were compared found be consistent. accurate valid evaluation, can evaluated