The purification, specificity, and role of dipeptidyl peptidase III inDictyostelium discoideum

Huang, Jui-Yen; Kim, Jun; Ramamurthy, Priya; Jones, Theodore H.D.

doi:10.1016/0147-5975(92)90016-k

Cited by 11 publications

(10 citation statements)

References 13 publications

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“…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: 95%

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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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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“…DPP III from human seminal plasma (10), lens tissue (11), and erythrocytes (12) has been purified and biochemically characterized, as were the enzymes from several other mammalian tissues, including bovine pituitary (13), rat brain (9), and liver (8). Regarding non-mammals, DPP III has been partially purified and characterized from the slime mold Dictyostelium discoideum (14) and Drosophila melanogaster (15).…”

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The First Structure of Dipeptidyl-peptidase III Provides Insight into the Catalytic Mechanism and Mode of Substrate Binding

Baral

Jajčanin-Jozić

Deller

et al. 2008

Journal of Biological Chemistry

111

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Dipeptidyl-peptidases III (DPP III) are zinc-dependent enzymes that specifically cleave the first two amino acids from the N terminus of different length peptides. In mammals, DPP III is associated with important physiological functions and is a potential biomarker for certain types of cancer. Here, we present the 1.95-Å crystal structure of yeast DPP III representing the prototype for the M49 family of metallopeptidases. It shows a novel fold with two domains forming a wide cleft containing the catalytic metal ion. DPP III exhibits no overall similarity to other metallopeptidases, such as thermolysin and neprilysin, but zinc coordination and catalytically important residues are structurally conserved. Substrate recognition is accomplished by a binding site for the N terminus of the peptide at an appropriate distance from the metal center and by a series of conserved arginine residues anchoring the C termini of different length substrates.

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The purification, specificity, and role of dipeptidyl peptidase III inDictyostelium discoideum

Cited by 11 publications

References 13 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

The First Structure of Dipeptidyl-peptidase III Provides Insight into the Catalytic Mechanism and Mode of Substrate Binding

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