| PEPD | |||||||||||||||||||||||||||||||||||||||||||||||||||
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| Aliases | PEPD, PROLIDASE, peptidase D | ||||||||||||||||||||||||||||||||||||||||||||||||||
| External IDs | OMIM:613230;MGI:97542;HomoloGene:239;GeneCards:PEPD;OMA:PEPD - orthologs | ||||||||||||||||||||||||||||||||||||||||||||||||||
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| Wikidata | |||||||||||||||||||||||||||||||||||||||||||||||||||
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Xaa-Pro dipeptidase, also known asprolidase, is anenzyme that in humans is encoded by thePEPDgene.[5][6][7]Prolidase is an enzyme in humans that plays a crucial role in protein metabolism and collagen recycling through the catalysis of the rate-limiting step in these chemical reactions.[8] This enzyme is coded by the gene PEPD (peptidase D), located on chromosome 19.[9]Serum prolidase activity is also currently being explored as abiomarker for diseases.
Xaa-Pro dipeptidase is a cytosolicdipeptidase that hydrolyzes dipeptides withproline orhydroxyproline at the carboxy terminus (but not Pro-Pro). It is important in collagen metabolism because of the high levels ofimino acids.[7] Mutations at the PEPD locus causeprolidase deficiency. This is characterised by Iminodipeptidurea,skin ulcers,mental retardation and recurrent infections.
Serum prolidase falls into the category of proteases, specifically exopeptidases. These EC numbers range from 3.4.11 to 3.4.19.[10]
Prolidases fall under a subclass ofmetallopeptidases that involve binuclearactive sitemetal clusters.[11] This metal cluster facilitatescatalysis by serving as asubstratebinding site, activatingnucleophiles, and stabilizing thetransition state. Furthermore, prolidases are classified under a smaller family called "pita-bread" enzymes, which cleaveamido-,imido-, andamidino- containing bonds.[12] The "pita-bread" fold, containing a metal center flanked by two well-defined substrate binding pockets enabled prolidase to specifically cleave between any non-prolineamino acid and proline.
The first ever solved structure of prolidase came from thehyperthermophilicarchaeonPyrococcus furiosus (Pfprol).[11] This dimer has a crystal structure shows two approximately symmetricalmonomers that both have anN-terminal domain, made up of a six-stranded mixed β-sheet flanked by fiveα-helices, a helical linker, andC-terminal domain, consisting of a mixed six-strandedβ-sheet flanked by four α-helices. The curved β-sheet of Domain II has a "pita-bread" fold. The active site lies on the inner surface of the β-sheet of Domain II, with a notable dinuclearCo cluster anchored by the side chains of twoaspartateresidues (Asp209 and Asp220), twoglutamate residues (Glu313 and Glu327), and ahistidine residue (His284).Carboxylate groups of aspartate andglutamine residues serve as bridges between the two Co atoms. In thecrystallization process, the Co atoms are replaced withZn, which hinders enzymatic activity.
Sequencehomology between human and Pfprol yield only 25% identity and 43% similarity.[13]
Two 493 amino acid chains construct serum prolidase, held together with C2 symmetry.[14] This C2 symmetry refers to the molecule's two-fold rotational symmetry without mirror symmetry.[15] Simply put, if serum prolidase were to be rotated at a 180º angle, it would look the same, however, it does not look the same in a mirror image. Furthermore, this structure has two domains: the N-terminal domain and the C-terminal domain, the latter of which carries the active site in the amino acid residues 185-493.[14] The active site is the area on the enzyme to which the substrate binds and catalysis occurs. This C-terminal domain has the ability to covalently bond to other prolidase enzymes to create a tetramer through disulfide bonds.[14] This domain performs a "pita-bread" fold, consisting of a bimetallic active center held together by two ɑ-helices and one antiparallel β-sheet.[14] Prolidase enzyme is considered homodimeric, meaning it is formed by two identical polypeptide chains.[8] There are both hydrophilic and hydrophobic residues in this enzyme, distributed evenly throughout.
Manganese ions (Mn2+) are utilized by serum prolidase as co-factors. Research into the crystal structure has found that two Mn2+ ions are required for the catalytic activity of this enzyme.[8] This requirement leads to prolidase being deemed a metal-activated peptidase, a term used to describe enzymes that catalyze the hydrolysis reaction changing peptides into amino acids having increased ability through the existence of metal ions. It has been indicated that one Mn2+ ion is tightly bound to His370, while the second is loosely bound to Asp276.[8]
Human prolidase has four crystal structures, HsProl-Mn, HsProl-Na-GlyPro, HsProl-Mg-LeuPro, and HsProl-Mn-Pro.[14] The first of these structures, HsProl-Mn, pertains to the activity of serum prolidase before binding the substrate.[14] Furthermore, HsProl-Na-GlyPro results from substrate degradation caused by the exchange of the Mn2+ ion with Na+. This is caused by the substrate GlyPro binding to the enzyme.[14] The third crystal structure of serum prolidase is HsProl-Mg-LeuPro. This structure functions similarly to HsProl-Na-GlyPro; however, the substrate utilized in this structure is LeuPro. Additionally, Mn2+ is replaced by Mg2+.[14] These differences cause the structure to be more stable with a lower turnover rate.[14] The final crystal structure of serum prolidase is HsProl-Mn-Pro, which employs Pro as the substrate.[14] This Pro comes from the reaction being catalyzed by this enzyme.[14] The crystal structure of prolidase is well-researched and recorded in the Protein Data Bank.[16]
The role of prolidase in human physiology is collagen breakdown. Collagen, the most prevalent protein in the human body, is necessary for maintaining strong connective tissues, cellular proliferation, and wound healing, among other functions.[17] As collagen is degraded, dipeptides are released as a byproduct. Serum prolidase absorbs and digests these byproducts so they can be reused in collagen production.[8] Proline is required for collagen production, further indicating the necessity of serum prolidase, as proline is a product of the prolidase reaction.[8] Wound healing is a paramount function in maintaining good health of the human body. Collagen uses its rigid properties to structurally support wounds and speed up the healing process.[8] As the wound heals, type III collagen is produced by fibroblasts, which is later replaced by type II collagen, then type I collagen.[8] These changes indicate different stages of the wound-healing process.
Due to proline's cyclic structure, only fewpeptidases could cleave the bond between proline and other amino acids.[18] Along withprolinase, prolidase are the only known enzymes that can break down dipeptides to yield free proline. Prolidase serve to hydrolyze both dietary andendogenous Xaa-Pro dipeptides. More specifically, it is essential in catalyzing the last step of the degradation of procollagen,collagen, and other proline-containing peptides into free amino acids to be used for cellular growth.[19] Additionally, it also participates in the process of recycling proline from Xaa-Pro dipeptides for collagen resynthesis. Proline and hydroyxyproline make up a quarter of the amino acid residues in collagen, which is the most abundant protein in the body by mass and plays an important role in maintainingconnective tissue in the body.[19][20]
Biochemical and structural analyses ofaminopeptidase (APPro),methionine aminopeptidase (MetAP), and prolidase, all members of the "pita-bread"metalloenzymes, suggest that they share a common mechanism scheme.[12] The main difference arises in the location of thecarbonyl oxygen atom of thescissile peptide bond.
The following mechanism shows a proposed scheme for a metal-dependent "pita-bread" enzyme with residue numbering corresponding to those found in methionine aminopeptidase fromE. coli.[12] As shown in Intermediate I of the figure, three potentialacidic amino acid residues interact with the N-terminus of the substrate in a fashion that is yet to be determined. The carbonyl and amide groups of the scissile peptide bond interact with the first metal ion, M1, in addition to His178 and His79, respectively. M1 and Glu204 activate a water molecule to prepare itnucleophilic attack at the carbonyl carbon of the scissile peptide bond. Then, thetetrahedralintermediate (Intermediate II) becomes stabilized from interactions with M1 and His178. Lastly, Glu204 donates aproton to the amine of the leavingpeptide (P1'). This leads to the breakdown of the intermediate (Intermediate III), which retains its interactions with M1 and His178.
The reaction pathway of prolidase is a fairly complicated process with many components involved. After a proton is removed from the bridge between the two Mn2+ ions, the GlyPro substrate causes a conformational change as it binds to the active site.[8] This GlyPro is held in place by hydrogen bonds formed by multiple amino acids in this structure.[8] The Gly-N atom of the GlyPro substrate and the Gly-O atom of the peptide bond each interact with the Mn2+ ions, which are stabilized by additional amino acids, leading to polarization.[8] This reaction causes the carbonyl carbon atom located on the peptide bond to receive a positive charge, which then reacts with the hydroxide ion formed by the Mn2+ ions, creating a tetrahedral intermediate.[8] A conformational change occurs, releasing the initial product glycine while the protein is still closed. An additional conformation change from closed to open occurs when proline, the final product of this reaction, is released.[8]
Post-translational modifications of prolidase regulate its enzymatic abilities.Phosphorylation of prolidase has been shown to increase its activity whiledephosphorylation leads to a decrease in enzyme activity.[21] Analysis of knownconsensus sequence required forserine/threonine phosphorylation revealed that prolidase contains at least three potential sites for serine/threonine phosphorylation. Nitric oxide, bothexogenously acquired andendogenously generated, was shown to increase prolidase activity in a time- anddose-dependent manner via phosphorylation at these serine and threonine sites.[22] Additionally, prolidase may also be regulated attyrosine phosphorylation sites, which are mediated byFAK andMAPKsignaling pathways.[21]
The presence of serum prolidase in the blood is a good indicator of the presence and severity of many types of diseases. For instance, Type 2 Diabetes mellitus patients have elevated levels of serum prolidase.[8] This is expected because high blood glucose leads to a decrease in collagen production and inflammatory cell generation, which depreciates wound healing ability.[8] Furthermore, Rheumatoid Arthritis, Ankylosing spondylitis, and benign joint hypermobility syndrome have corresponded with low serum prolidase levels.[8] Prolidase has become a prominent marker of cancer progression in patients with various types of cancer. Depending on the elevated levels of serum prolidase in the blood, physicians are able to determine tumor size, stage of cancer, and prognosis, all of which help significantly in treating these diseases.[8]
Analysis of serum prolidase levels has been used to detect the severity of liver disease in some instances.[8] Research has indicated the correlation between chronic liver diseases and serum prolidase. For instance, one study suggested an increase in serum prolidase levels during the initial stages of cirrhotic liver fibrosis, followed by a decrease as the disease progressed.[8] Moreover, analysis of serum prolidase levels in alcoholic hepatitis patients has displayed higher levels compared to cirrhosis patients.[8]
Serum prolidase is a highly necessary enzyme in the human body. Through its many functions, most notably collagen recycling, prolidase is widely used in the overall metabolism of humans. The reaction pathway of this enzyme is key in regulating the synthesis and degradation of collagen, a vital protein necessary for multiple aspects of the body. Irregular levels of serum prolidase in the blood are indicative of various diseases and conditions in humans.
Deficiency in prolidase leads to a rare, severeautosomal recessive disorder (prolidase deficiency) that causes many chronic, debilitating health conditions in humans.[23] Thesephenotypical symptoms vary and may includeskin ulcerations,mental retardation,splenomegaly, recurrentinfections,photosensitivity,hyperkeratosis, and unusual facial appearance. Furthermore, prolidase activity was found to be abnormal compared to healthy levels in various medical conditions including but limited to:bipolar disorder,breast cancer,endometrial cancer,keloid scar formation,erectile dysfunction,liver disease,lung cancer,hypertension,melanoma, andchronic pancreatitis.[18] In some cancers with increased levels of prolidase activity, such as melanoma, the differential expression of prolidase and its substrate specificity for dipeptides with proline at thecarboxyl end suggests the potential of prolidase in becoming a viable, selectiveendogenous enzyme target for prolineprodrugs.[24]Serum prolidase enzyme activity is also currently being explored as a possible, reliablemarker for diseases includingchronic hepatitis B andliver fibrosis.[25][26][27]
Decontamination: Prolidase from the hyperthermophilic archaeon Pyrococcus furiosus (Pfprol) shows potential for application in decontamination oforganophosphorusnerve agents inchemical warfare agents.[28] Additionally, prolidase could also serve to detectfluorine-containing organophosphorusneurotoxins, like the G-type chemical warfare agents, and couldantagonize organophosphorousintoxication and protect against the effects ofdiisopropylfluorophosphate whenencapsulated inliposomes.[29][30]
