Summary for peptidase M03.005: peptidyl-dipeptidase Dcp

Summary Alignment Tree Sequences Sequence features Distribution Structure Literature Substrates Inhibitors


MEROPS Namepeptidyl-dipeptidase Dcp
Other namesdipeptidyl carboxypeptidase
Name and HistoryFormerly, peptidases able to cleave C-terminal dipeptides from peptide substrates were named peptidyl-dipeptide hydrolase. In 1972, IUB recognized only one activity of this type, the mammalian angiotensin I-converting enzyme (ACE, EC ACE is known to hydrolyze angiotensin I and bradykinin, peptides involved in the regulation of blood pressure (Corvol et al., 1995). In 1978, an enzyme from Escherichia coli with catalytic properties very similar to those of ACE (Yaron, 1976) was included into the same entry (EC, which then was renamed dipeptidyl carboxypeptidase. The E. coli enzyme was moved to a new entry (EC in 1981 and renamed peptidyl-dipeptidase B, to distinguish it from mammalian peptidyl-dipeptidase A. The bacterial enzyme was also called dipeptidyl carboxypeptidase II in the period 1984-1988, and in the period 1989-1992 the term peptidyl dipeptidase II was recommended. In 1992, entry EC was closed and reincluded into EC as peptidyl dipeptidase A, including enzymes of both prokaryotic and eukaryotic origin until 1996. On the basis of recent sequencing studies (Hamilton & Miller, 1992, Henrich et al., 1993), the enzymes from E. coli and Salmonella typhimurium have again been separated out and transferred to the new entry They are now referred to as peptidyl-dipeptidase Dcp.
Domain architecture
MEROPS Classification
Classification Clan MA >> Subclan MA(E) >> Family M3 >> Subfamily A >> M03.005
Holotypepeptidyl-dipeptidase Dcp (Escherichia coli), Uniprot accession P24171 (peptidase unit: 2-681), MERNUM MER0001158
History Identifier created: Handbook of Proteolytic Enzymes (1998) Academic Press, London.
Catalytic typeMetallo
PeplistIncluded in the Peplist with identifier PL00140
NC-IUBMBSubclass 3.4 (Peptidases) >> Sub-subclass 3.4.15 (Peptidyl-dipeptidases) >> Peptidase
EnzymologyBRENDA database
PreparationDcp has been purified from E. coli B (1200-fold) (Yaron, 1976) and from S. typhimurium, in which dcp was 50-fold overexpressed from a plasmid (Conlin & Miller, 1995). In E. coli K-12, the enzyme was enriched 80-fold from transformants carrying a recombinant low copy-number plasmid variant (Henrich et al., 1993). Dcp from E. coli (Cunha et al., 2009) and Leishmania donovani (Goyal et al., 2006) have also been expressed with a C-terminal His-tag and purified in a Nickel-column with high efficiency. Dcp has also been purified to homogeneity from Pseudomonas sp. WO24 with similar properties to E. coli enzyme, but the sequence has not been determined (Ogasawara et al., 1997).
SpecificityDcp is a carboxydipeptidase that removes dipeptides from the free C-termini of unprotected tetrapeptides, higher peptides and N-blocked tripeptides. Dcp is unable to cleave peptides containing Pro at P1' position, between two Gly residues, with blocked C-terminus or with a C-terminal D-amino acid (Yaron, 1976). Free tripeptides are not cleaved by Dcp. The enzyme hydrolyses the ACE substrates angiotensin I (Yaron et al., 1972), bradykinin and Hip-His-Leu (Yaron, 1976). In the 90s, Dcp activity was determined using Ac-Ala-Ala-Ala as substrate and the dipeptide Ala-Ala released was detected by HPLC and ninhydrin method respectively by (Hamilton & Miller, 1992) and (Conlin & Miller, 1995). Recently, using PS-SC fluorogenic libraries, the amino acid preferences of the E. coli Dcp subsites S3, S2, S1 and S1' were mapped(Cunha et al., 2009). It was seen that the S1 subsite has a strong preference for basic amino acids as Arg, which was the best-accepted residue in P1 position. The acidic residue Asp is not tolerated at this subsite. The S2 pocket of Dcp shows remarkable selectivity for substrates containing Phe at P2 position. The S3 subsite seems to be less important for enzyme activity, because it tolerated a broad range of amino acids. Peptides containing Lys and Arg at P1' position were very susceptible to hydrolysis by E. coli Dcp and the S1' subsite did not tolerate a Pro residue, as expected. The enzyme is slightly activated by manganese, magnesium, calcium and cobalt at 1 mM. Dcp from E. coli B (Yaron et al., 1972), but not from E. coli K-12 (Henrich et al., 1993), is activated by 0.05 mM of cobalt (5- to 8-fold). Activation by chloride anion or high salt concentration was not detected. Copper, nickel and zinc are potent inhibitors. No activity is detectable in the presence of 1,10-phenanthroline and EDTA has only a minor inhibitory effect.
pH optimumOptimum pH values of 8.2 and 7.5 have been determined for Dcp purified respectively from E. coli B (Yaron et al., 1972) and E. coli K-12 (Henrich et al., 1993), using Hip-His-Leu as substrate. A pI value of 5.2 has been determined for bacterial enzyme.
StructureDcp from E. coli and S. typhimurium share 79% sequence identity and has 680 amino acid residues with a molecular mass of 77.5 kDa (Henrich et al., 1993). Bacterial Dcp was placed in the M3 family due to its structural characteristics, including the presence of the potential zinc-binding signature HEXXH (Jongeneel et al., 1989), which is the distinguishing feature of the MA clan of metallopeptidases (Chu & Orlowski, 1985). Dcp from E. coli and S. typhimurium are quite similar (32% identity) to the oligopeptidase A enzymes of both bacterial species (Hamilton & Miller, 1992). Dcp crystallization was carried out in the presence of an octapeptide inhibitor, and the 2.0 Å was determined using multiple-wavelength anomalous diffraction (MAD) phasing techniques. Dcp crystal structure is similar to neurolysin and thimet oligopeptidase, other M3 peptidases, and presents a topology resembling to ACE. Dcp also displays a bi-lobed structure in its open conformation with the catalytic site located at the base of a deep channel attached to the internal surface of subdomain I. The strict dipeptidyl carboxypeptidase specificity of Dcp is explained by an Arg side-chain backed by a Glu residue, together with two Tyr phenolic groups that provide a charged anchor for fixing the C-terminal carboxylate group of the P2' residue of the bound substrate (Comellas-Bigler et al., 2005).
LocationThe enzyme properties indicate a cytoplasmic location, but recently about 10% of the Dcp activity was observed in the periplasmic space (Deutch & Soffer, 1978). Dcp could function in the breakdown of peptides provided in the medium, but most Dcp substrates seem to be poorly transported into the cell (Deutch & Soffer, 1978, Vimr & Miller, 1983). The major role of this enzyme appears to be the cleavage of intracellular proteins (Vimr et al., 1983).
PhysiologyLiberation of C-terminal dipeptides from intracellular oligopeptides.
Biological aspectsApart from the D-Ala-D-Ala carboxypeptidases, Dcp is the only C-terminal exopeptidase identified in E. coli or S. typhimurium. The dcp gene is transcribed as a single species of monocistronic mRNA in both bacterial species from well-identified promoters, and there is no evidence for regulation at the transcriptional level (Hamilton & Miller, 1992, Henrich et al., 1993). Dcp is present in less than 100 copies per cell, suggested by codon preferences. Post-translational modifications were not identified, apart from the removal of N-terminal N-formyl-Met (which may be incomplete for the S. typhimurium enzyme).
Distinguishing featuresDas & Soffer, 1976 observed that antibodies raised against rabbit ACE did not affect the activity of E. coli Dcp, suggesting a structural dissimilarity between these two carboxypeptidases. Recently, crystallization studies further support the proposed difference (Comellas-Bigler et al., 2005). Unlike Dcp, ACE is activated by chloride. Although Dcp and oligopeptidase A are capable of hydrolysing some of the same small peptide substrates, the specificity and inhibitor susceptibility of both enzymes are clearly discernible. Given the structural similarity and functional differences exhibited by Dcp and oligopeptidase A, it is absolutely inadequate to evaluate the properties of similar enzymes purely based on amino acid similarity. Therefore, the assignment of some predicted proteins, such as a putative oligopeptidase of Haemophilus influenzae (Fleischmann et al., 1995) and a potential metalloendopeptidase (MEP) of Schizophyllum commune (Stankis et al., 1992), which share significant similarity with Dcp, remains unclear without the support of biochemical data.
Contributing authorsThaysa Paschoalin, Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de São Paulo, Rua Botucatu, 862, 8° andar, 04023-062 São Paulo, SP, Brazil.