Steady-State Kinetics of Glutamine Cyclotransferase

Gololobov, Mikhail Yu.; Song, In‐Seok; Wang, W.Y.; Bateman, Robert C.

doi:10.1006/abbi.1994.1117

Cited by 30 publications

(34 citation statements)

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“…We previously noted that the N-terminal amino acid sequence of PQC exhibits no homology with the amino acid sequences of the beef and human enzymes [11]. We, among others, have also observed that the mechanism of PQC glutamine cyclase catalysis is different from that of mammalian glutamine cyclases [7,11,13]. The findings reported here thus support the hypothesis that PQC and mammalian glutamine cyclases belong to distinct families of enzymes, and have only the capacity to catalyse cyclization of L-glutaminyl-peptides in common.…”

Section: Discussionmentioning

confidence: 99%

“…In addition, increasing the temperature of PQC solutions up to 95°C does not induce any conformational transition. Finally, some residual native-like structure still persists in the presence of 7 M guanidinium hydrochloride [7,11,13]. The structural cause of this remarkable stability was investigated by analysis of the circular dichroism and infrared spectra of the enzyme, its hydrogen-deuterium exchange characteristics, and by its susceptibility to proteolysis by trypsin and chymotrypsin.…”

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confidence: 99%

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Papaya glutamine cyclase, a plant enzyme highly resistant to proteolysis, adopts an all‐β conformation

Oberg

Ruysschaert

Azarkan

et al. 1998

European Journal of Biochemistry

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Glutamine cyclases catalyse the conversion of L-glutaminyl-peptides into 5-oxoprolyl-peptides with the concomitant liberation of ammonia. We report here biophysical characterisation of the glutamine cyclase present in the laticiferous cells of the plant Carica papaya. After purification to near homogeneity, this enzyme was subjected to limited proteolysis and found to exhibit a high resistance to degradation and nicking. The structural reasons for this property were examined using circular dichroism and infrared spectroscopies. By combining the analyses of the infrared and CD spectra of papaya glutamine cyclase, its susceptibility to proteolysis, and its hydrogen-deuterium exchange characteristics, we conclude that this protein contains extensive β-sheet structure and is likely to have only short immobile loops connecting its β-strands.Keywords : secondary structure ; circular dichroism; infrared spectroscopy ; limited proteolysis; hydrogendeuterium exchange.The aerial parts of the tropical plant Carica papaya contain laticiferous cells which constitute a particularly rich source of cysteine proteinases. The well-documented proteinases papain, glycyl endopeptidase, chymopapain and caricain are all secreted by these cells [1Ϫ4]. Besides these proteinases, minor protein constituents including two chitinases [5,6], a glutamine cyclase [7] and a serine proteinase inhibitor [8] have been isolated and partially characterised.Although they are synthesised as inactive preproenzymes, the four papaya proteinases exist in their mature forms in the laticifers [9] because freshly collected papaya latex exhibits full proteolytic activity. The catalytic competence of papain-like cysteine proteinases requires the generation of a catalytic-site imidazolium-thiolate ion pair and the protonic dissociation of a carboxylic acid function with a pKa value close to 4 [10], which controls the ion pair geometry. Three elements help sustain full proteolytic activity in the papaya laticiferous cells: (a) the absence of cysteine proteinase inhibitors, (b) the presence of a high concentration (about 20 mM) of low molecular-mass thiol-containing compounds, and (c) a buffering capacity that maintains the pH around 5.2. From a physiological point of view, papaya laticifers may therefore be regarded as an extreme milieu. The structural reasons for the ability of the latex enzymes to elude -A-benzoyl-DL-arginine-p-nitroanilide; BTPNA, N-A-benzoyl-DL-tyrosine-p-nitroanilide. Enzymes. Glutamine cyclases (EC 2.3.2.5); papain (EC 3.4.22.2) ; glycyl endopeptidase (EC 3.4.22.25); chymopapain (EC 3.4.22.6) ; caricain (EC 3.4.22.30). rapid in vivo degradation through autolysis and proteolysis has not previously been examined.Within this context, we present a study of a recently purified glutaminyl-peptide cyclotransferase from the laticifers of C. papaya (referred to here as papaya glutamine cyclase, or PQC). This enzyme catalyses the conversion of L-glutaminyl-peptides into 5-oxoprolyl-peptides with concomitant liberation of ammonia [11], as shown...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Papaya glutamine cyclase, a plant enzyme highly resistant to proteolysis, adopts an all‐β conformation

Oberg

Ruysschaert

Azarkan

et al. 1998

European Journal of Biochemistry

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“…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.…”

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confidence: 99%

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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“…The assay consisted of the chromogenic substrate Gln-pNA (1 mM), pyroglutamyl aminopeptidase (0.25 U), and an appropriate amount of QC in a final volume of 0.25 ml buffer (0.05 M Tricine/NaOH, pH 8.0). This pH was reported to be within the optimal range for the catalysis of both QC and pyroglutamyl aminopeptidase (9,15). Due to the composition of the storage buffer of pyroglutamyl aminopeptidase, assay mixtures contained cysteamine (0.1 mM), sodium chloride (2 mM), EDTA (0.1 mM), and 2% (v/v) glycerol.…”

Section: Assaysmentioning

confidence: 99%