Zechel D L, Konermann L, Withers S G, Douglas D J
Department of Chemistry, University of British Columbia, Vancouver, Canada.
Biochemistry. 1998 May 26;37(21):7664-9. doi: 10.1021/bi980445o.
For the first time, the new technique of time-resolved electrospray ionization mass spectrometry (ESI-MS) has been used to accurately measure the pre-steady state kinetics of an enzymatic reaction by monitoring a transient enzyme intermediate. The enzyme used to illustrate this approach, Bacillus circulans xylanase, is a retaining glycosidase that hydrolyzes xylan or beta-xylobiosides through a double-displacement mechanism involving a covalent xylobiosyl-enzyme intermediate. A low steady state level of this intermediate formed during the hydrolysis of 2,5-dinitrophenyl beta-d-xylobioside was detected by time-resolved ESI-MS. The low concentration of this intermediate and its rate of formation did not permit pre-steady state kinetic analysis. By contrast, the covalent intermediate accumulates fully when the Tyr80Phe mutant hydrolyzes the same substrate. Using time-resolved ESI-MS, the pre-steady state kinetic parameters for the formation of the covalent intermediate in the mutant xylanase have been determined. The kinetic data are in agreement with those determined by monitoring the release of 2, 5-dinitrophenol with stopped-flow UV-vis spectroscopy. This demonstrates that time-resolved ESI-MS can be used to accurately monitor the pre-steady state kinetics of enzymatic reactions, with the advantage of identifying transient enzyme intermediates by their mass.
首次将时间分辨电喷雾电离质谱（ESI-MS）新技术用于通过监测瞬态酶中间体来精确测量酶促反应的预稳态动力学。用于说明该方法的酶——环状芽孢杆菌木聚糖酶，是一种保留型糖苷酶，它通过涉及共价木二糖基 - 酶中间体的双置换机制水解木聚糖或β - 木二糖苷。通过时间分辨ESI-MS检测到在2,5 - 二硝基苯基β - D - 木二糖苷水解过程中形成的该中间体的低稳态水平。该中间体的低浓度及其形成速率不允许进行预稳态动力学分析。相比之下，当Tyr80Phe突变体水解相同底物时，共价中间体会完全积累。使用时间分辨ESI-MS，已确定突变体木聚糖酶中共价中间体形成的预稳态动力学参数。动力学数据与通过停流紫外 - 可见光谱监测2,5 - 二硝基苯酚释放所确定的数据一致。这表明时间分辨ESI-MS可用于精确监测酶促反应的预稳态动力学，其优点是通过质量鉴定瞬态酶中间体。
