Pi Na, Leary Julie A
Department of Chemistry, University of California at Berkeley, Berkeley, California 94720, USA.
J Am Soc Mass Spectrom. 2004 Feb;15(2):233-43. doi: 10.1016/j.jasms.2003.10.009.
The traditional method used to investigate the reaction specificity of an enzyme with different substrates is to perform individual kinetic measurements. In this case, a series of varied concentrations are required to study each substrate and a non-regression analysis program is used several times to obtain all the specificity constants for comparison. To avoid the large amount of experimental materials, long analysis time, and redundant data processing procedures involved in the traditional method, we have developed a novel strategy for rapid determination of enzyme substrate specificity using one reaction system containing multiple competing substrates. In this multiplex assay method, the electrospray ionization mass spectrometry (ESI-MS) technique was used for simultaneous quantification of multiple products and a steady-state kinetics model was established for efficient specificity constant calculation. The system investigated was the bacterial sulfotransferase NodH (NodST), which is a host specific nod gene product that catalyzes the sulfate group transfer from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to natural Nod factors or synthetic chitooligosaccharides. Herein, the reaction specificity of NodST for four chitooligosaccharide acceptor substrates of different chain length (chitobiose, chitotriose, chitotetraose, and chitopentaose) was determined by both individual kinetic measurements and the new multiplex ESI-MS assay. The results obtained from the two methods were compared and found to be consistent. The multiplex ESI-MS assay is an accurate and valid method for substrate specificity evaluation, in which multiple substrates can be evaluated in one assay.
用于研究酶与不同底物反应特异性的传统方法是进行单独的动力学测量。在这种情况下，研究每种底物都需要一系列不同的浓度，并且要多次使用非回归分析程序来获得所有特异性常数以进行比较。为了避免传统方法中涉及的大量实验材料、较长的分析时间和冗余的数据处理程序，我们开发了一种新策略，使用包含多种竞争性底物的一个反应体系来快速测定酶的底物特异性。在这种多重测定方法中，采用电喷雾电离质谱（ESI-MS）技术同时对多种产物进行定量，并建立了稳态动力学模型以高效计算特异性常数。所研究的体系是细菌磺基转移酶NodH（NodST），它是一种宿主特异性的nod基因产物，催化硫酸基团从3'-磷酸腺苷5'-磷酸硫酸酯（PAPS）转移到天然Nod因子或合成的壳寡糖上。在此，通过单独的动力学测量和新的多重ESI-MS测定法，确定了NodST对四种不同链长的壳寡糖受体底物（壳二糖、壳三糖、壳四糖和壳五糖）的反应特异性。比较了两种方法获得的结果，发现它们是一致的。多重ESI-MS测定法是一种准确有效的底物特异性评估方法，可在一次测定中评估多种底物。
