Evidence for Essential Histidines in Human Pituitary Glutaminyl Cyclase

Bateman, Robert C.; Temple, Jeffrey S.; Misquitta, Stephanie A.; Booth, Rachell E.

doi:10.1021/bi011177o

Cited by 29 publications

(48 citation statements)

References 26 publications

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“…1C), suggesting some roles in catalysis. Mutations of those amino acids decreased enzyme activity significantly (Table 1), in agreement with some results by Bateman et al (32). Interestingly, the acidic E201, D248, and D305 are pointing to each other at pH 6.5 and 8.0, likely forming hydrogen bonds between them.…”

Section: Resultssupporting

confidence: 90%

Crystal structures of human glutaminyl cyclase, an enzyme responsible for protein N-terminal pyroglutamate formation

Huang

Liu

Cheng

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

107

120

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N-terminal pyroglutamate (pGlu) formation from its glutaminyl (or glutamyl) precursor is required in the maturation of numerous bioactive peptides. The aberrant formation of pGlu may be related to several pathological processes, such as osteoporosis and amyloidotic diseases. This N-terminal cyclization reaction, once thought to proceed spontaneously, is greatly facilitated by the enzyme glutaminyl cyclase (QC). To probe this important but poorly understood modification, we present here the structure of human QC in free form and bound to a substrate and three imidazole-derived inhibitors. The structure reveals an ␣͞␤ scaffold akin to that of two-zinc exopeptidases but with several insertions and deletions, particularly in the active-site region. The relatively closed active site displays alternate conformations due to the different indole orientations of Trp-207, resulting in two substrate (glutamine t-butyl ester)-binding modes. The single zinc ion in the active site is coordinated to three conserved residues and one water molecule, which is replaced by an imidazole nitrogen upon binding of the inhibitors. Together with structural and kinetic analyses of several active-site-mutant enzymes, a catalysis mechanism of the formation of protein N-terminal pGlu is proposed. Our results provide a structural basis for the rational design of inhibitors against QC-associated disorders.crystallography ͉ intramolecular cyclization ͉ posttranslational modification ͉ Alzheimer's disease ͉ aminopeptidase N -terminal pyroglutamate (pGlu) formation from its glutaminyl precursor is an important posttranslational or cotranslational event in the processing of numerous bioactive neuropeptides, hormones, and cytokines during their maturation in the secretory pathway. These regulatory peptides require the N-terminal pGlu to develop the proper conformation for binding to their receptors and͞or to protect the N termini of the peptides from exopeptidase degradation (1, 2). Previously, this Nterminal cyclization reaction was thought to proceed spontaneously, until the glutaminyl cyclases (QCs) were identified as catalysts that are responsible for this posttranslational modification (3, 4).QCs (EC 2.3.2.5) are acyltransferases that have been identified in both animal and plant sources (3-5). They are abundant in mammalian neuroendocrine tissues, such as hypothalamus and pituitary (4, 6), and are highly conserved from yeast to human. Animal QCs were shown to have distinct structure and protein stability from plant QCs despite their similar molecular masses [i.e., 33-40 kDa (5, 7)]. No bacterial QCs have been reported thus far; however, the mammalian QCs were predicted to exhibit remarkable homology to the bacterial double-zinc aminopeptidases (8, 9).In humans, several genetic diseases, such as osteoporosis, a multifactorial hormonal disease that is characterized by reduced bone mass and microarchitectural deterioration of bone tissue (10), appear to result from mutations of the QC gene. The gene encoding QC (QPCT) lies on chromosome 2p22.3. ...

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Section: Resultssupporting

confidence: 90%

Crystal structures of human glutaminyl cyclase, an enzyme responsible for protein N-terminal pyroglutamate formation

Huang

Liu

Cheng

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

107

120

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“…5), the binding motif His-Asp-Glu-Asp-His of the two Zn 2ϩ ions present in this clan of hydrolases is also conserved in human QC. Furthermore, as shown in another study (9), modification of two of the identified histidine residues, His-140 and His-330, which are probably necessary for metal binding (Fig. 5), leads to a complete loss of catalytic activity.…”

Section: Figsupporting

confidence: 66%

“…Because of the complete reactivation by the addition of Zn 2ϩ ions to apo QC, one can conclude that human and probably all mammalian QCs are Zn 2ϩ -dependent. Recently, a relationship of the tertiary structure of human QC and the aminopeptidase from Vibrio proteolyticus, a prominent member of the clan MH family M28 of metallopeptidases, was proposed (9). Comparing the sequence of human QC with those of two members of the clan MH (Fig.…”

Section: Figmentioning

confidence: 99%

“…In the case of human QC, these residues are the putative counterparts to the peptidases. The shaded histidines (His-140 and His-330) indicate residues that were identified as being essential for QC catalysis (9). attacking water molecule and polarize the scissile bond, making it susceptible to nucleophilic attack during transition state formation, with its progression to and the subsequent collapse of the tetrahedral intermediate followed by amid bond cleavage (20).…”

Section: Figmentioning

confidence: 99%

“…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).…”

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confidence: 95%

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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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Glutaminyl-peptide cyclotransferase

Springer Handbook of Enzymes

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Evidence for Essential Histidines in Human Pituitary Glutaminyl Cyclase

Cited by 29 publications

References 26 publications

Crystal structures of human glutaminyl cyclase, an enzyme responsible for protein N-terminal pyroglutamate formation

Crystal structures of human glutaminyl cyclase, an enzyme responsible for protein N-terminal pyroglutamate formation

Identification of Human Glutaminyl Cyclase as a Metalloenzyme

Glutaminyl-peptide cyclotransferase

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