Isolation, Catalytic Properties, and Competitive Inhibitors of the Zinc-Dependent Murine Glutaminyl Cyclase

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• 作者：Stephan Schilling ; Holger Cynis ; Alex von Bohlen ; Torsten Hoffmann ; Michael Wermann ; Ulrich Heiser ; Mirko Buchholz ; Katrin Zunkel ; and Hans-Ulrich Demuth
• 刊名：Biochemistry
• 出版年：2005
• 出版时间：October 11, 2005
• 年：2005
• 卷：44
• 期：40
• 页码：13415 - 13424
• 全文大小：231K
• 年卷期：v.44,no.40(October 11, 2005)
• ISSN：1520-4995

文摘

Murine glutaminyl cyclase (mQC) was identified in the insulinoma cell line -TC 3 bydetermination of enzymatic activity and RT-PCR. The cloned cDNA was expressed in the secretory pathwayof 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 virtuallyidentical 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 K_i value of42 ±2 M. 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 andthe substrate is necessary in order to bind to the active site of the enzyme. In contrast to imidazole andcysteamine, the heterocyclic chelators 1,10-phenanthroline, 2,6-dipicolinic acid, and 8-hydroxyquinolineinactivated 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 bytotal reflection X-ray fluorescence spectrometry and atomic absorption spectroscopy in mQC revealedstoichiometric amounts of zinc bound to the protein. Metal ion depletion appeared to have no significanteffect on protein structure as shown by fluorescence spectroscopy, suggesting a catalytic role of zinc. Theresults 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 mainlybased on a loss of one metal binding site.