Purification and Characterization of L-Pyrrolidonecarboxylate Peptidase from Bacillus amyloliquefaciens

Tsuru, Daisuke; Fujiwara, Katsuo; Kado, Kunio

doi:10.1093/oxfordjournals.jbchem.a132148

Cited by 50 publications

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“…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. In some cases, assay buffer also contained up to 5 mM EDTA, which has been shown to stabilize the auxiliary enzyme (15). K i values of QC inhibition were not influenced by EDTA.…”

Section: Methodsmentioning

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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Section: Methodsmentioning

confidence: 99%

Identification of Human Glutaminyl Cyclase as a Metalloenzyme

Schilling

Niestroj

Rahfeld

et al. 2003

Journal of Biological Chemistry

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“…Carboxypeptidase Y and aminopeptidase M digestions were carried out in 0.1 M pyridinium acetate at 37°C and pH 6.0 for 1.5 h and at 37°C and pH 7.0 for 1 h, respectively. 5-Oxoprolyl peptidase digestion was done as described by Tsuru et al [29].…”

Section: Enzymatic Digestionmentioning

confidence: 99%

“…7), it gave a signal at of H20, the results suggest that the N-terminus of this peptide is a 5-oxoproline (pyrrolidone carboxylic acid) residue (Glp). Moreover, when peptide V-5 was digested with 5-oxoprolyl peptidase [29] and the digest was measured by FAB mass spectrometry (Fig. 7), the signal of the peptide at m/z = 944.…”

Section: Numbers Of Amino Acid Residues In Parentheses Indicate Neamentioning

confidence: 99%

Insulin-stimulating protein from human plasma. Chemical characteristics and biological activity

et al. 1986

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A protein that potentiates the action of insulin in vitro was purified from human plasma. When reduced with 2-mercaptoethanol and then carboxymethylated, it yielded a single subunit, indicating that it was composed of two identical subunits connected by a single disulfide bond. This modified subunit tended to inhibit rather than stimulate insulin activity. A distinctive feature of the amino acid composition of this protein (H-ISP) was the absence of histidine, arginine, and tryptophan. The molecular mass, subunit composition, the characteristic amino acid composition and the N-terminal amino acid residue of H-ISP are very similar to those of human plasma apolipoprotein A-I1 (apo A-11). The isoelectric point of H-ISP was estimated to be 4.91, which is identical with that of the major apo A-I1 isoform.H-ISP did not itself have insulin-like activity in increasing C 0 2 liberation from labeled glucose and 2-deoxyglucose uptake by isolated rat adipocytes, but it potentiated the action of insulin in these parameters. It had no appreciable affect on the binding or degradation of 12'I-labeled insulin by adipocytes. Like H-ISP, apo A-I1 isolated from human plasma also had no insulin-like activity by itself, but stimulated the effect of insulin on COa production from labeled glucose in isolated rat adipocytes. From these results, it is concluded that H-ISP is identical with the major apo A-I1 isoform. Incubation of isolated adipocytes with H-ISP resulted in marked increase in the activity of pyruvate dehydrogenase in a dose-dependent manner in the absence of added insulin. H-ISP also stimulated pyruvate dehydrogenase activity in a subcellular system consisting of plasma membranes and mitochondria from rat adipocytes. The effect of H-ISP on pyruvate dehydrogenase activity could be produced by treatment of the isolated mitochondria1 fraction alone.

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“…PGP-1 was first isolated from Bacillus amyloliquefaciens, and its enzymatic properties were characterized by us (2). We have also reported the cloning and sequencing of the enzyme gene (3).…”

mentioning

confidence: 99%

The Mechanism of Substrate Recognition of Pyroglutamyl-peptidase I from Bacillus amyloliquefaciens as Determined by X-ray Crystallography and Site-directed Mutagenesis

Itō

Inoue

Takahashi

et al. 2001

Journal of Biological Chemistry

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Pyroglutamyl-peptidase is able to specifically remove the amino-terminal pyroglutamyl residue protecting proteins or peptides from aminopeptidases. To clarify the mechanism of substrate recognition for the unique structure of the pyrrolidone ring, x-ray crystallography and site-directed mutagenesis were applied. The crystal structure of pyroglutamyl-peptidase bound to a transition state analog inhibitor (Inh), pyroglutaminal, was determined. Two hydrogen bonds were located between the main chain of the enzyme and the inhibitor (71:O⅐⅐⅐H-N:Inh and Gln71:N-H⅐⅐⅐OE:Inh), and the pyrrolidone ring of the inhibitor was inserted into the hydrophobic pocket composed of Phe-10, Phe-13, Thr-45, Ile-92, Phe-142, and Val-143. To study in detail the hydrophobic pocket, Phe-10, Phe-13, and Phe-142 were selected for mutation experiments. The k cat value of the F10Y mutant decreased, but the two phenylalanine mutants F13Y and F142Y did not exhibit significant changes in kinetic parameters compared with the wild-type enzyme. The catalytic efficiencies (k cat /K m ) for the F13A and F142A mutants were less than 1000-fold that of the wild-type enzyme. The x-ray crystallographic study of the F142A mutant showed no significant change except for a minor one in the hydrophobic pocket compared with the wild type. These findings indicate that the molecular recognition of pyroglutamic acid is achieved through two hydrogen bonds and an insertion in the hydrophobic pocket. In the pocket, Phe-10 is more important to the hydrophobic interaction than is Phe-142, and furthermore Phe-13 serves as an "induced fit" mechanism.

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Purification and Characterization of L-Pyrrolidonecarboxylate Peptidase from Bacillus amyloliquefaciens

Cited by 50 publications

References 0 publications

Identification of Human Glutaminyl Cyclase as a Metalloenzyme

Identification of Human Glutaminyl Cyclase as a Metalloenzyme

Insulin-stimulating protein from human plasma. Chemical characteristics and biological activity

The Mechanism of Substrate Recognition of Pyroglutamyl-peptidase I from Bacillus amyloliquefaciens as Determined by X-ray Crystallography and Site-directed Mutagenesis

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