Presence of a dipeptidyl aminopeptidase III in Saccharomyces cerevisiae.

Watanabe, Yasuhiro; Kumagai, Yuka; Fujimoto, Yukio

doi:10.1248/cpb.38.246

Cited by 22 publications

(24 citation statements)

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“…Since the latter inhibitors affect a variety of serine and cysteine peptidases, further studies are needed to assign the DAP to one of four classes of active site structure. Furthermore, while Co(II) inhibited the DAP activity, it stimulated mammalian and yeast DAP III to hydrolyze enkephalin and Arg-Arg-␤NA, respectively (27). These results show that the purified DAP from Pseudomonas sp.…”

Section: Discussionmentioning

confidence: 72%

“…These properties (optimum pH and molecular mass) of the DAP appeared to have some resemblance to those of mammalian and S. cerevisiae DAP III rather than to those of mammalian DAP I. However, the enzyme differs from DAP III with respect to inhibitor sensitivity (27). The enzyme is not inhibited by EDTA or thiol, carboxy, and general serine protease inhibitors (DFP and PMSF), but it is inhibited by TLCK, chymostatin, and leupeptin.…”

Section: Discussionmentioning

confidence: 87%

“…DAP I and II have not been found in microorganisms, except for recent reports that Bacteroides ruminicola (19) and Dictyostelium discoideum (4) may possess DAP I-like activities capable of hydrolyzing Gly-Arg-4-methoxy-␤NA (Gly-Arg-MNA) and Ala-Ala-Ala-Ala and hydrolyzing Gly-Arg-4-methylcoumarinamide (Gly-Arg-MCA), respectively. Concerning other types of DAPs, DAP III was found to be present only in Saccharomyces cerevisiae (27) and D. discoideum (7). Although these enzymes have been shown to resemble mammalian DAP III, some differences were noted and their molecular nature remains obscure.…”

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confidence: 99%

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A novel dipeptidyl aminopeptidase from Pseudomonas sp. strain WO24

et al. 1996

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An activity similar to that of dipeptidyl aminopeptidase I (DAP I) which releases dipeptide from Gly-Argp-nitroanilide (Gly-Arg-pNA) was detected in a Pseudomonas sp. An enzyme was isolated and purified about 400-fold by a series of column chromatographies. The enzyme, named DAP BI (DAP from bacteria, type I), was revealed to be homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing. The molecular mass was estimated to be 82 kDa by SDS-PAGE and 65 kDa by gel filtration, suggesting that the enzyme may be a monomer. The enzyme had an isoelectric point of 4.7. It is optimally active at pH 9.0. The K m and V max of the enzyme for Gly-Arg-pNA were 0.25 mM and 195 mol/min/mg, respectively. The purified enzyme did not hydrolyze Gly-Phe-pNA, which was also a substrate for DAP I, whereas it hydrolyzed Arg-Arg-4-methoxy-␤-naphthylamide (Arg-Arg-MNA), a model substrate for DAP III. The K m and V max for Arg-Arg-MNA were 0.019 mM and 145 mol/min/mg, respectively. This purified enzyme can also catalyze the removal of Asp-Arg from the N termini of angiotensins I and II. The enzyme activity was completely inhibited by Zn(II) (0.5 mM), tosyl-L-Lys-chloromethyl ketone (0.1 mM), and leupeptin (0.1 mM) and partially inhibited by Co(II) (0.5 mM) and chymostatin (0.1 mM), whereas the enzyme was not affected by general serine protease inhibitors (phenylmethylsulfonyl fluoride and diisopropylfluorophosphate) and thiol protease inhibitors. The substrate specificity, classification of catalytic site, and other enzymatic properties demonstrate that this enzyme is distinct from the previously described mammalian DAPs I and III and Saccharomyces cerevisiae DAP III. These results indicate that DAP BI may be a new type of the DAP family.Dipeptidyl aminopeptidase (DAP) is capable of sequentially removing dipeptides from the amino termini of peptides and proteins. In mammalian cells, four distinct enzymes with DAP activity have been purified from the liver, brain, and pituitary on the basis of substrate specificity by using unique naphthylamide (NA) substrates, specific pH optima, and catalytic classification (13, 15). DAP I (cathepsin C) cleaves Gly-Arg-␤-naphthylamide (Gly-Arg-␤NA) and Gly-Phe-␤NA at pH 6, and DAP II cleaves Lys-Ala-␤NA with a pH optimum of 5. DAP III degrades Arg-Arg-␤NA with a pH optimum of 8 to 9, while DAP IV hydrolyzes Gly-Pro-␤NA at pH 8. DAP I is a lysosomal cysteine protease belonging to the papain superfamily. It possesses a broad specificity (20) for liberating dipeptides whose amino-terminal amino acid is not basic (Arg or Lys) and whose penultimate amino acid is not proline and that are not linked to proline through a peptide bond. DAP I appears to play an important role not only in intracellular protein degradation, but also in cell growth and processing and activation of several enzymes (18). On the other hand, DAP III shows relatively narrow substrate specificity on dipeptide NAs but a wide range of activity on peptide substrates. In particular, DAP III pre...

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Section: Discussionmentioning

confidence: 72%

Section: Discussionmentioning

confidence: 87%

mentioning

confidence: 99%

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A novel dipeptidyl aminopeptidase from Pseudomonas sp. strain WO24

et al. 1996

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“…In these reports, the enzymes were not purified to homogeneity, and their characteristics were not clarified. DAP III-like enzymes were discovered in Saccharomyces cerevisiae (22) and D. discoideum (3,4,7). Although these enzymes have been shown to resemble mammalian DAP III, some differences were noted and their molecular nature remains obscure.…”

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confidence: 99%

Two types of novel dipeptidyl aminopeptidases from Pseudomonas sp. strain WO24

et al. 1996

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Two kinds of dipeptidyl aminopeptidase I (DAP I [cathepsin C])-like activities which hydrolyzeGly-Phe-pnitroanilide (Gly-Phe-pNA) were detected in Pseudomonas sp. strain WO24. They were purified and characterized. The isolated enzymes, named DAP BII and DAP BIII, were revealed to be homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing. DAP BII was estimated to have a molecular mass of 150,000 Da by gel filtration and a subunit size of 73,000 Da by SDS-PAGE, indicating it to be a homodimer. The molecular mass of DAP BIII was evaluated to be approximately 60,000 Da by gel filtration and 69,000 Da by SDS-PAGE, indicating that it is monomeric. The isoelectric points of DAP BII and DAP BIII were 6.1 and 5.0, and their optimal pHs were 8.0 and 8.5 to 9.0, respectively. The result of peptide mapping for DAP BII and DAP BIII showed that these enzymes consist of different components. Both enzymes were completely inhibited by diisopropylphosphofluoride but not by general thiol inhibitors, indicating that they are serine proteases. DAP BII and DAP BIII hydrolyzed Gly-Phe-pNA but not Gly-Arg-pNA, both of which are model substrates for mammalian DAP I. Despite these shared activities toward DAP I, DAP BII released dipeptides from Ala-Ala-pNA and Lys-Ala-4-methylcoumarinamide (a substrate for DAP II), whereas DAP BIII did not hydrolyze either of these compounds and was presumed to prefer substrates composed of bulky, hydrophobic amino acids at P1 and P1 positions. In addition, DAP BII showed no endopeptidase activity, whereas DAP BIII possessed the activity on N-terminally blocked peptide derivatives besides exopeptidase activity. Assays performed with bioactive peptides such as angiotensin I and neuromedin N as substrates indicate that DAP BII has a considerably broader substrate specificity than DAP BIII and is able to hydrolyze an X-Pro bond, an imido bond that few peptidases and no known DAPs can cleave. These characteristics, namely, substrate specificities, molecular mass, pI, peptide mapping, pH optimum, and effect of inhibitors, suggested that the two DAPs purified in this work are distinct enzymes and do not belong to any of the previously reported DAP classes.

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“…5,10 The same type of enzyme was partially purified and described from two lower eukaryotes, the slime mold Dictyostelium discoideum and the yeast Saccharomyces cerevisiae. 12,13 The physiological substrates of DPP III are mostly unknown, although it hydrolyzes a number of biologically active peptides in vitro. Besides its contribution in the final steps of intracellular protein catabolism, a regulatory role for DPP III was suggested.…”

Section: Introductionmentioning

confidence: 99%

Human Dipeptidyl Peptidase III: the Role of Asn406 in Ligand Binding and Hydrolysis

et al. 2011

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Abstract. Human dipeptidyl peptidase III (DPP III) is a member of the zinc-metallopeptidase family M49 with a role in intracellular protein catabolism. Ligand-free crystal structure of yeast and human orthologues provided insight into the enzyme's active center and enabled some assumptions about the enzymesubstrate interactions. The molecular modeling performed for human and yeast DPP III indicated involvement of Asn406 and Asn415, respectively in ligand binding. To investigate further the role of this asparagine residue, conserved in all eukaryotic M49 peptidases, a site-directed mutagenesis of human DPP III was carried out. Replacement of Asn406 with Gln, but not with Ser, resulted in a large decrease in the enzyme's binding affinity for competitive peptide inhibitor and in catalytic efficiency. Molecular dynamics simulations performed for enzyme-substrate complexes, revealed changed coordination of the active-site zinc ion and the orientation of the catalytically important His568, in N406Q, but not in the N406S complexes.(doi: 10.5562/cca1808)

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Presence of a dipeptidyl aminopeptidase III in Saccharomyces cerevisiae.

Cited by 22 publications

References 0 publications

A novel dipeptidyl aminopeptidase from Pseudomonas sp. strain WO24

A novel dipeptidyl aminopeptidase from Pseudomonas sp. strain WO24

Two types of novel dipeptidyl aminopeptidases from Pseudomonas sp. strain WO24

Human Dipeptidyl Peptidase III: the Role of Asn406 in Ligand Binding and Hydrolysis

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