Selevsek Nathalie, Tholey Andreas, Heinzle Elmar, Liénard Benoit M R, Oldham Neil J, Schofield Christopher J, Heinz Uwe, Adolph Hans-Werner, Frère Jean-Marie
Department of Biochemical Engineering, Saarland University, Saarbrücken, Germany.
Chemistry Research Laboratory, University of Oxford, Mansfield Road, OX1 3TA, Oxford, UK.
J Am Soc Mass Spectrom. 2006 Jul;17(7):1000-1004. doi: 10.1016/j.jasms.2006.03.010. Epub 2006 Jul 1.
Metallo-β-lactamases (MBLs) are targets for medicinal chemistry as they mediate bacterial resistance to β-lactam antibiotics. Electrospray-ionization mass spectrometry (ESI-MS) was used to study the inhibition by a set of mercaptocarboxylates of two representative MBLs with different optimal metal stoichiometries for catalysis. BcII is a dizinc MBL (Class B1), whilst the CphA MBL (Class B2) exhibits highest activity with a single zinc ion in the active site. Experimental parameters for the detection of the metallo-enzyme and the metallo-enzyme-inhibitor complexes were evaluated and optimized. Following investigations on the stoichiometry of metal binding, the affinity of the inhibitors was investigated by measuring the relative abundance of the complex compared to the metalloprotein. The results for the BcII enzyme were in general agreement with solution assays and demonstrated that the inhibitors bind to the dizinc form of the BcII enzyme. The results for the CphA(Zn) complex unexpectedly revealed an increased affinity for the binding of a second metal ion in the presence of thiomandelic acid. The results demonstrate that direct ESI-MS analysis of enzyme:inhibitor complexes is a viable method for screening inhibitors and for the rapid assay of the enzyme:metal:inhibitor ratios.
金属β-内酰胺酶（MBLs）是药物化学的研究靶点，因为它们介导细菌对β-内酰胺抗生素的耐药性。采用电喷雾电离质谱（ESI-MS）研究了一组巯基羧酸盐对两种具有不同最佳催化金属化学计量比的代表性MBLs的抑制作用。BcII是一种双锌MBL（B1类），而CphA MBL（B2类）在活性位点含有单个锌离子时表现出最高活性。对金属酶和金属酶-抑制剂复合物检测的实验参数进行了评估和优化。在研究金属结合化学计量后，通过测量复合物相对于金属蛋白的相对丰度来研究抑制剂的亲和力。BcII酶的结果与溶液分析基本一致，表明抑制剂与BcII酶的双锌形式结合。CphA(Zn)复合物的结果出乎意料地显示，在硫代扁桃酸存在下，对第二种金属离子结合的亲和力增加。结果表明，酶-抑制剂复合物的直接ESI-MS分析是筛选抑制剂和快速测定酶-金属-抑制剂比例的可行方法。
