Gao Hong, Leary Julie A
Department of Chemistry, University of California at Berkeley, Berkeley, California 94720-1460, USA.
J Am Soc Mass Spectrom. 2003 Mar;14(3):173-81. doi: 10.1016/S1044-0305(02)00867-X.
An electrospray ionization mass spectrometry based assay was developed for kinetic measurements and inhibitor screening of yeast hexokinase. There is considerable discrepancy in the literature as to the accuracy of kinetic data obtained for hexokinase. In the assay described herein, the product, glucose 6-phosphate was directly monitored by ion trap mass spectrometry and quantified using an internal standard, 2 deoxy-glucose 6-phosphate. The kinetic parameters, K(M) and V(max) for the two substrates were determined without using a coupling enzyme as is normally employed in the traditional spectrophotometric assay for systems lacking a chromophore. In addition, hexokinase was successfully immobilized onto an amino-link gel, and a mock library was screened against the immobilized enzyme for the identification of possible inhibitors. After comparing the mass spectra of the library before and after incubation, trehalose 6-phosphate, ADP, and oxidized glutathione were differentiated from other weak or non-inhibitors. Inhibition behavior of ADP with respect to ATP was further evaluated with the ESI-MS assay and the value of K(i) was determined. This ESI-MS assay was demonstrated to be both accurate and precise for determining kinetic constants and for identifying enzyme inhibitors.
开发了一种基于电喷雾电离质谱的分析方法，用于酵母己糖激酶的动力学测量和抑制剂筛选。关于己糖激酶动力学数据的准确性，文献中存在相当大的差异。在本文所述的分析方法中，通过离子阱质谱直接监测产物6-磷酸葡萄糖，并使用内标2-脱氧-6-磷酸葡萄糖进行定量。在不使用传统分光光度法中通常用于缺乏发色团系统的偶联酶的情况下，测定了两种底物的动力学参数K(M)和V(max)。此外，己糖激酶成功固定在氨基连接凝胶上，并针对固定化酶筛选了模拟文库以鉴定可能的抑制剂。比较文库孵育前后的质谱后，区分了6-磷酸海藻糖、ADP和氧化型谷胱甘肽与其他弱抑制剂或非抑制剂。用ESI-MS分析方法进一步评估了ADP对ATP的抑制行为，并测定了K(i)值。结果表明，这种ESI-MS分析方法在测定动力学常数和鉴定酶抑制剂方面既准确又精确。
