Schilling Stephan, Cynis Holger, von Bohlen Alex, Hoffmann Torsten, Wermann Michael, Heiser Ulrich, Buchholz Mirko, Zunkel Katrin, Demuth Hans-Ulrich
Probiodrug AG, Weinbergweg 22, 06120 Halle/Saale, Germany.
Biochemistry. 2005 Oct 11;44(40):13415-24. doi: 10.1021/bi051142e.
Murine glutaminyl cyclase (mQC) was identified in the insulinoma cell line beta-TC 3 by determination of enzymatic activity and RT-PCR. The cloned cDNA was expressed in the secretory pathway of the methylotrophic yeast Pichia pastoris and purified after fermentation using a new three-step protocol. mQC converted a set of various substrates with very similar specificity to human QC, indicating a virtually identical catalytic competence. Furthermore, mQC was competitively inhibited by imidazole derivatives. A screen of thiol reagents revealed cysteamine as a competitive inhibitor of mQC bearing a Ki value of 42 +/-2 microM. Substitution of the thiol or the amino group resulted in a drastic loss of inhibitory potency. The pH dependence of catalysis and inhibition support that an uncharged nitrogen of the inhibitors and the substrate is necessary in order to bind to the active site of the enzyme. In contrast to imidazole and cysteamine, the heterocyclic chelators 1,10-phenanthroline, 2,6-dipicolinic acid, and 8-hydroxyquinoline inactivated mQC in a time-dependent manner. In addition, citric acid inactivated the enzyme at pH 5.5. Inhibition by citrate was abolished in the presence of zinc ions. A determination of the metal content by total reflection X-ray fluorescence spectrometry and atomic absorption spectroscopy in mQC revealed stoichiometric amounts of zinc bound to the protein. Metal ion depletion appeared to have no significant effect on protein structure as shown by fluorescence spectroscopy, suggesting a catalytic role of zinc. The results demonstrate that mQC and probably all animal QCs are zinc-dependent catalysts. Apparently, during evolution from an ancestral protease, a switch occurred in the catalytic mechanism which is mainly based on a loss of one metal binding site.
通过酶活性测定和逆转录聚合酶链反应（RT-PCR），在胰岛素瘤细胞系β-TC 3中鉴定出小鼠谷氨酰胺环化酶（mQC）。克隆的互补DNA（cDNA）在甲基营养型酵母毕赤酵母的分泌途径中表达，并在发酵后使用新的三步方案进行纯化。mQC对一组各种底物的催化特异性与人类QC非常相似，表明其催化能力几乎相同。此外，mQC受到咪唑衍生物的竞争性抑制。对硫醇试剂的筛选显示，半胱胺是mQC的竞争性抑制剂，其抑制常数（Ki）值为42±2微摩尔。硫醇或氨基的取代导致抑制效力急剧丧失。催化和抑制的pH依赖性表明，抑制剂和底物的不带电荷的氮对于结合到酶的活性位点是必要的。与咪唑和半胱胺不同，杂环螯合剂1,10-菲咯啉、2,6-二吡啶甲酸和8-羟基喹啉以时间依赖性方式使mQC失活。此外，柠檬酸在pH 5.5时使该酶失活。在锌离子存在下，柠檬酸的抑制作用被消除。通过全反射X射线荧光光谱法和原子吸收光谱法对mQC中的金属含量进行测定，结果显示与该蛋白质结合的锌的化学计量量。如荧光光谱所示，金属离子耗竭似乎对蛋白质结构没有显著影响，这表明锌具有催化作用。结果表明，mQC以及可能所有动物的QC都是锌依赖性催化剂。显然，在从祖先蛋白酶进化的过程中，催化机制发生了转变，这主要是由于一个金属结合位点的丧失。
