Continuous Spectrometric Assays for Glutaminyl Cyclase Activity

Schilling, Stephan; Hoffmann, Torsten; Wermann, Michael; Heiser, Ulrich; Wasternack, Claus; Demuth, Hans‐Ulrich

doi:10.1006/abio.2001.5560

Cited by 52 publications

(57 citation statements)

References 21 publications

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“…Papaya QC was purified from crude papain as described previously (14). All other chemicals were of analytical or high pressure liquid chromatography grade.…”

Section: Methodsmentioning

confidence: 99%

“…QC Assays-QC activity was assayed essentially as described elsewhere (14). Briefly, the assay reactions (250 l) consisted of varying concentrations (ϳ0.25-4 K m ) of H-Gln-AMC or H-Gln-Gln-OH in 0.05 M Tris-HCl, pH 8.0.…”

Section: Methodsmentioning

confidence: 99%

“…pH Dependence-For the investigation of the pH dependence of QC catalysis and inhibition, the previously developed fluorometric assay was used (14). Determinations were carried out at 30°C in a buffer originally used by Ellis and Morrison consisting of 0.06 M acetic acid, 0.06 M Mes, and 0.12 M Tris (16).…”

Section: Methodsmentioning

confidence: 99%

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Identification of Human Glutaminyl Cyclase as a Metalloenzyme

Schilling

Niestroj

Rahfeld

et al. 2003

Journal of Biological Chemistry

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Glutaminyl cyclases (QC) 1 (EC 2.3.2.5) are acyltransferases present in animals and plants that catalyze the conversion of N-terminal glutaminyl residues into pyroglutamic acid with the concomitant liberation of ammonia (Scheme 1). Several peptide hormones and proteins carry N-terminal pyroglutamyl residues. Previously, the formation of N-terminal pyroglutamate from glutamine was assumed to proceed spontaneously (1). However, the QCs were identified more recently as catalysts of the reaction in both mammals and plants (2-5). Generally, QCs from both mammalian and plant sources appear to be very similar monomeric proteins that are expressed in the secretory pathways and have similar molecular masses, ϳ33 and ϳ40 kDa, respectively (6, 7). Their primary structures, however, reveal no sequence homology, and the enzymes feature completely different folding patterns. Whereas plant QC consists almost solely of ␤-sheet structure, mammalian QCs are predicted to possess an ␣/␤-fold (8 -10). Furthermore, plant QC does not share sequence or structural homology to other plant enzymes, belonging, apparently, to a separate enzyme subfamily (4). Mammalian QCs, however, exhibit remarkable homology toward bacterial aminopeptidases, suggesting their evolutionary origin in this protein family (9).Investigating the substrate specificity of both enzymes, we recently found differences between papaya and human QC (24). Although the catalysis of cyclization of and the inhibition by modified N-terminal residues were quite different, both enzymes catalyzed the cyclization of oligopeptides with similar specificity rate constants. Moreover, they also catalyzed the formation of a five-membered lactam ring from L-␤-homoglutaminyl peptides. Based on the present state of knowledge, it is assumed that plant and animal QCs catalyze the formation of N-terminal pyroglutamic acid (pGlu) residues by enabling the intramolecular cyclization of the glutaminyl residue in a noncovalent manner (11,12). However, the results of the substrate specificity study revealed differences in substrate recognition by both enzymes.Thus, a more detailed analysis of the inhibition pattern of plant and human QCs should help to deepen our understanding of QC catalysis. To date, however, systematic inhibitor data exist only for plant QC, which is competitively inhibited by peptides containing an N-terminal proline residue (13), whereas human QC was shown to be inactivated by 1,10-phenanthroline and reduced 6-methylpterin (3). Thus, we investigated the inhibiting potency of heterocyclic compounds from different structural classes. Among them, imidazole and structurally related compounds were found to be the most efficient competitive inhibitors of human QC. The data provide the first insights into enzyme/inhibitor interactions, offer clues for further optimization of the inhibitory structure, and reveal novel aspects of human QC catalysis.

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“…Papaya QC was purified from crude papain as described previously (14). All other chemicals were of analytical or high pressure liquid chromatography grade.…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Identification of Human Glutaminyl Cyclase as a Metalloenzyme

Schilling

Niestroj

Rahfeld

et al. 2003

Journal of Biological Chemistry

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“…The respective 1-alkylimidazol moiety was introduced by means of watersoluble carbodiimide (WSCD). The nitrovinyldiamines (22)(23)(24)(25)(26)(27)(28)(29)(30)(41)(42)(43)(44)(45), and 52) were obtained by a stepwise reaction of 2,2-bis-methylthio-1-nitroethene with the corresponding amines (Scheme 5). 33 The synthesis of the 2-thioxopyrimidin-4(1H)-ones (31-35, 46-50, and 53) was accomplished via the corresponding (5-alkyloxycarbonyl)arylthiourea derivatives by the formation of an amide bond leading to a ring closure at the 1-nitrogen of the thiourea moiety (Scheme 6).…”

Section: Resultsmentioning

confidence: 99%

Inhibitors for Human Glutaminyl Cyclase by Structure Based Design and Bioisosteric Replacement

Buchholz¹,

Hamann²,

Aust³

et al. 2009

J. Med. Chem.

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The inhibition of human glutaminyl cyclase (hQC) has come into focus as a new potential approach for the treatment of Alzheimer's disease. The hallmark of this principle is the prevention of the formation of Abeta(3,11(pE)-40,42), as these Abeta-species were shown to be of elevated neurotoxicity and likely to act as a seeding core leading to an accelerated formation of Abeta-oligomers and fibrils. Starting from 1-(3-(1H-imidazol-1-yl)propyl)-3-(3,4-dimethoxyphenyl)thiourea, bioisosteric replacements led to the development of new classes of inhibitors. The optimization of the metal-binding group was achieved by homology modeling and afforded a first insight into the probable binding mode of the inhibitors in the hQC active site. The efficacy assessment of the hQC inhibitors was performed in cell culture, directly monitoring the inhibition of Abeta(3,11(pE)-40,42) formation.

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“…The progress of the reaction also has been monitored by measurement of the product by reversed-phase high-performance liquid chromatography (HPLC) with spectrophotometric and fluorimetric detection using [Gln 1 ] LH-RH and Gln-Leu-Tyr-Glu-Asn-Lys-ε-(Dns)-OH, respectively [5,6]. Commonly used assays of this enzyme activity also include a spectrophotometric method to measure the ammonia released during cyclization of the dipeptide substrate Gln-Gln [16] and a continuous spectrometric assay using the chromogenic substrate Gln-p-nitroanilide, and fluorogenic substrates Gln-2-naphthylamide and Gln-4-methylcoumarinylamide [17].…”

Section: Introductionmentioning

confidence: 99%