Isolation, catalytic properties, and competitive inhibitors of the zinc-dependent murine glutaminyl cyclase.
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.
Schilling
Stephan
S
Probiodrug AG, Weinbergweg 22, 06120 Halle/Saale, Germany.
Cynis
Holger
H
von Bohlen
Alex
A
Hoffmann
Torsten
T
Wermann
Michael
M
Heiser
Ulrich
U
Buchholz
Mirko
M
Zunkel
Katrin
K
Demuth
Hans-Ulrich
HU
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*
D018076:DNA, Complementary; D007093:Imidazoles; D011994:Recombinant Proteins; D019343:Citric Acid; C029899:imidazole; D019881:Aminoacyltransferases; C059280:glutaminyl-peptide cyclotransferase; D015032:Zinc; D009584:Nitrogen