title: Isolation, catalytic properties, and competitive inhibitors of the zinc-dependent murine glutaminyl cyclase.
abstract: 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.
authors.affiliation: Probiodrug AG, Weinbergweg 22, 06120 Halle/Saale, Germany.
authors.forename: Stephan
authors.initials: S
authors.lastname: Schilling
authors.forename: Holger
authors.initials: H
authors.lastname: Cynis
authors.forename: Alex
authors.initials: A
authors.lastname: von Bohlen
authors.forename: Torsten
authors.initials: T
authors.lastname: Hoffmann
authors.forename: Michael
authors.initials: M
authors.lastname: Wermann
authors.forename: Ulrich
authors.initials: U
authors.lastname: Heiser
authors.forename: Mirko
authors.initials: M
authors.lastname: Buchholz
authors.forename: Katrin
authors.initials: K
authors.lastname: Zunkel
authors.forename: Hans-Ulrich
authors.initials: HU
authors.lastname: Demuth
mesh_terms: D000595:Amino Acid Sequence; D019881:Aminoacyltransferases / Q000737:chemistry*; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D002417:Cattle; D019343:Citric Acid / Q000494:pharmacology; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D019143:Evolution, Molecular; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007093:Imidazoles / Q000737:chemistry; D007700:Kinetics; D051379:Mice; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009584:Nitrogen / Q000737:chemistry; D017785:Photons; D010843:Pichia / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013379:Substrate Specificity; D013997:Time Factors; D015032:Zinc / Q000737:chemistry*
chemical_list: D018076:DNA, Complementary; D007093:Imidazoles; D011994:Recombinant Proteins; D019343:Citric Acid; C029899:imidazole; D019881:Aminoacyltransferases; C059280:glutaminyl-peptide cyclotransferase; D015032:Zinc; D009584:Nitrogen