Aminopeptidase

Crystal structure of the open state of human endoplasmic reticulum aminopeptidase 1 ERAP1[1]
Identifiers
SymbolPeptidase_M1
PfamPF01433
MEROPSM1
OPM superfamily227
OPM protein3mdj
CDDcd09595
Membranome534
Available protein structures:
Pfam  structures / ECOD  
PDBRCSB PDB; PDBe; PDBj
PDBsumstructure summary

Aminopeptidases are enzymes that catalyze the cleavage of amino acids from the amino terminus (N-terminus) of proteins or peptides (exopeptidases). "They are widely distributed throughout the animal and plant kingdoms and are found in many subcellular organelles, in cytosol, and as membrane components. Aminopeptidases are used in essential cellular functions. Many, but not all, of these peptidases are zinc metalloenzymes."[2]

Aminopeptidases occur in both soluble and membrane-bound forms and can be found in various cellular compartments as well as in the extracellular environment.[3] Their broad substrate specificity allows them to remove N-terminal amino acids from almost all unsubstituted oligopeptides.[4] For instance, Aminopeptidase N (AP-N) is particularly abundant in the brush border membranes of kidney, small intestine, and placenta, and is also rich in liver.[4] AP-N is involved in the final digestion of peptides generated from hydrolysis of proteins by gastric and pancreatic proteases.[5]

Some aminopeptidases are monomeric, and others are assemblies of relatively high mass (50 kDa) subunits. cDNA sequences are available for several aminopeptidases and a crystal structure of the open state of human endoplasmic reticulum Aminopeptidase 1 ERAP1 is presented here.[1] "Amino acid sequences determined directly or deduced from cDNAs indicate some amino acid sequence homologies in organisms as diverse as Escherichia coli and mammals, particularly in catalytically important residues or in residues involved in metal ion binding."[2]

One important aminopeptidase is a zinc-dependent enzyme produced and secreted by glands of the small intestine. It helps the enzymatic digestion of proteins. Additional digestive enzymes produced by these glands include dipeptidases, maltase, sucrase, lactase, and enterokinase.[6]

History[edit]

The discovery and characterization of aminopeptidases date back to the early 20th century. In 1929, Linderstrøm-Lang and Sato first introduced the term "aminopeptidase" to describe enzymes that cleave amino acids from the N-terminus of peptides.[7] Since then, numerous aminopeptidases have been identified and studied, revealing their diverse functions and applications.

In the 1950s and 1960s, the discovery of leucine aminopeptidase (LAP) and aminopeptidase N (APN) marked important milestones in the field. LAP was found to be crucial for protein digestion, while APN was recognized for its role in the regulation of peptide-mediated effects.[4][8] These discoveries were pivotal in understanding the physiological functions of aminopeptidases and their involvement in health and disease.

The subsequent decades saw extensive research into the structure, function, and mechanisms of action of various aminopeptidases. For example, the M1 family of aminopeptidases, which includes puromycin-sensitive aminopeptidase (PSA), was characterized by conserved zinc-dependent sites and exopeptidase motifs.[8] The study of PSA in different model organisms revealed its essential roles in growth and behavior, and mutations in psa orthologs were linked to meiotic errors and reduced embryonic viability.[8] Aminopeptidase N, also known as AP-N or CD13, was extensively characterized for its broad substrate specificity and its presence in various tissues such as the brush border membranes of the kidney, small intestine, and placenta.[4] The enzyme's role in brain function and its identification as the human cluster differentiation antigen CD13 on the surface of myeloid cells further highlighted its biological significance.

The characterization of these enzymes has not only advanced our understanding of their biological roles but has also opened up new avenues for therapeutic interventions. The discovery of selective inhibitors for enzymes like ERAP1, which is involved in antigen presentation, exemplifies the potential for targeting aminopeptidases in drug development.[9]

Structure and classification[edit]

Aminopeptidases are a diverse group of enzymes that play crucial roles in various biological processes, including protein digestion, cell growth, and immune response. They are classified based on their substrate specificity and catalytic mechanism into two main categories: metalloaminopeptidases and cysteine aminopeptidases.

Metalloaminopeptidases[edit]

Metalloaminopeptidases require metal ions, such as zinc or manganese, for their catalytic activity. These enzymes are characterized by a conserved HEXXH motif in their active site, which is involved in metal ion coordination. This motif is crucial for the catalytic function of the enzyme, as the histidine residues within the motif coordinate the metal ion, facilitating the hydrolysis of the peptide bond.[10] Metalloaminopeptidases are the largest and most homogenous class of aminopeptidases, with over 35 families identified within the MA clan according to the MEROPS database. This classification is based on structural similarities and evolutionary relationships, indicating a common ancestral origin for these enzymes,[10] Examples of metalloaminopeptidases include aminopeptidase N (APN), leucine aminopeptidase (LAP), and aminopeptidase A (APA).[11][12]

Cysteine aminopeptidase[edit]

Cysteine aminopeptidases, on the other hand, rely on a catalytic cysteine residue for their activity. These enzymes are part of a broader group known as cysteine proteases, which share a common catalytic mechanism involving a nucleophilic cysteine thiol in a catalytic triad or dyad. The catalytic triad typically consists of cysteine, histidine, and aspartate residues, where the cysteine acts as a nucleophile, the histidine as a base, and the aspartate stabilizes the histidine.[13] Examples of cysteine aminopeptidases include cathepsin H and aminopeptidase B.[11]

Structural overview[edit]

The structure of aminopeptidases varies depending on the specific enzyme, but they generally consist of a catalytic domain and additional domains that contribute to substrate recognition and regulation. For instance, Aminopeptidase N (APN)/CD13, a type II metalloprotease, consists of 967 amino acids with a short N-terminal cytoplasmic domain, a single transmembrane part, and a large cellular ectodomain containing the active site.[11] The ectodomain is responsible for substrate binding and hydrolysis, highlighting the importance of domain organization in the function of aminopeptidases.[13]

Mechanisms[edit]

Aminopeptidases function as monozinc enzymes, where a zinc ion is crucial for their catalytic activity. The enzymatic mechanism involves the coordination of the zinc ion with water molecules and specific amino acid residues in the active site, enabling the hydrolysis of peptide bonds. Conserved residues and motifs within aminopeptidases significantly impact their catalytic efficiency and specificity. For instance, vertebrate aminopeptidases like aminopeptidase B contain evolutionarily conserved tyrosines essential for their enzymatic function. In aminopeptidase A (APA), residues Arg368 and Arg386 in the S2' subsite are vital for inhibitor binding and catalysis. These residues interact with APA inhibitors' P2' and P1' residues, enhancing inhibitory potency. The structural flexibility of aminopeptidases is critical for their catalytic mechanism. For example, ERAP1 displays distinct conformations, with domain IV's position relative to domain II affecting catalytic efficiency. Mutations disrupting interdomain interactions can reduce activity. IRAP exhibits active-site plasticity, accommodating various substrates through ligand-induced conformational changes that restrict solvent access to the catalytic center, showcasing its substrate permissiveness.

Production[edit]

Aminopeptidases are produced by a wide range of organisms, including bacteria, fungi, plants, and animals, and they serve essential roles in various biological processes.

Bacterial aminopeptidases[edit]

In bacteria, aminopeptidases are produced by both facultative and obligate strains and can be found in different cellular locations such as the cytoplasm, membranes, associated with the cell envelope, or secreted into the extracellular media.[14] These enzymes are involved in the catabolism of exogenously supplied peptides and are necessary for the final steps of protein turnover. They also participate in specific functions like the cleavage of N-terminal methionine from newly synthesized peptide chains (methionine aminopeptidases), stabilization of multicopy ColE1 based plasmids (aminopeptidase A), and the cleavage of N-terminal pyroglutamate (pyroglutamyl aminopeptidase.[14]

Fungal aminopeptidases[edit]

Fungi, particularly species like Aspergillus oryzae and Aspergillus sojae, produce aminopeptidases that have applications in the food industry as debittering agents.[15] These enzymes are also of interest for their potential biotechnological applications. For example, leucine aminopeptidase (LAP) from Aspergillus species has been found to be thermostable and could be used to control the degree of hydrolysis and flavor development in a wide range of substrates.[15]

Mammalian aminopeptidases[edit]

In mammals, aminopeptidases are produced in various tissues and organs, such as the liver, kidney, and intestine. They are involved in protein digestion, regulation of peptide-mediated effects, and the metabolism of bioactive peptides.[4] Aminopeptidase N (AP-N), also known as CD13, is particularly abundant in the brush border membranes of the kidney, small intestine, and placenta, and is also rich in the liver.[4] It has a broad substrate specificity and is involved in the final digestion of peptides generated from hydrolysis of proteins by gastric and pancreatic proteases.[4]

Uses and applications[edit]

Aminopeptidases have diverse applications across various fields such as biotechnology, medicine, and the food industry. Their ability to specifically target peptide bonds has made them invaluable in processes ranging from food production to therapeutic interventions.

Food industry[edit]

Debittering[edit]

In the food industry, aminopeptidases from Aspergillus oryzae and Aspergillus sojae are utilized for debittering protein hydrolysates, including those used in soy sauce and miso production. These enzymes help remove bitter-tasting peptides, enhancing the flavor and palatability of these products.[16]

Cheese ripening[edit]

Aminopeptidases play a crucial role in cheese ripening by participating in the proteolysis of milk proteins. This enzymatic action contributes significantly to the development of the cheese's flavor and texture, making aminopeptidases essential in the cheese-making process.[16]

Protein sequencing[edit]

Edman degrading[edit]

In protein sequencing, aminopeptidases are employed in the Edman degradation method. This technique involves the sequential removal and identification of the N-terminal amino acid of proteins, facilitating the elucidation of their amino acid sequence.[16]

Therapeutic targets[edit]

Hypertension[edit]

Aminopeptidase A (APA) is implicated in blood pressure regulation by converting angiotensin II to angiotensin III. APA inhibitors are being explored as potential antihypertensive agents, offering a novel approach to managing hypertension.[17]

Inflammation[edit]

Aminopeptidase N (APN) has been associated with the pathogenesis of inflammatory diseases such as rheumatoid arthritis and inflammatory bowel disease. Inhibitors of APN have demonstrated anti-inflammatory effects in animal models, positioning them as potential therapeutic agents for these conditions.[18]

Cancer[edit]

Several aminopeptidases, including APN, APA, and leucine aminopeptidase (LAP), are overexpressed in various cancers. Their involvement in tumor growth, invasion, and angiogenesis makes them attractive targets for cancer therapy. Aminopeptidase inhibitors have shown promise in preclinical studies as potential anticancer agents.[18]

Digestive processes[edit]

Protein digestion[edit]

Aminopeptidases in the gastrointestinal tract, such as APN and APA, are essential for the digestion of dietary proteins. They facilitate the absorption and utilization of amino acids by cleaving them from the N-terminus of peptides.[18]

Bioactive peptide metabolism[edit]

These enzymes also play a role in the metabolism of bioactive peptides, including hormones and growth factors. By regulating the levels of these peptides, aminopeptidases contribute to homeostasis and physiological process modulation.[18]

Biosensors and diagnostic tools[edit]

Biosensors development[edit]

Aminopeptidases have been utilized in creating biosensors for detecting specific amino acids or peptides. These biosensors generate a measurable signal in the presence of the target analyte, leveraging the catalytic activity of aminopeptidases.[18]

Diagnostic markers[edit]

The activity and expression levels of aminopeptidases have been explored as diagnostic markers for diseases like liver disorders and cancer. Variations in these parameters can indicate pathological conditions, aiding in disease diagnosis and monitoring.[18]

Safety and storage[edit]

Optimal storage conditions for aminopeptidases[edit]

Aminopeptidases require specific storage conditions to maintain their stability and enzymatic activity. For instance, human aminopeptidase A is stable at a pH range of 7.0-8.5 and can be stored at -20°C for several months without significant loss of activity. Similarly, a halotolerant intracellular protease from Bacillus subtilis strain FP-133, which exhibits aminopeptidase activity, retains full activity after being stored in 7.5% (w/v) NaCl at 4°C for 24 hours.[19] These examples indicate that aminopeptidases generally require neutral pH conditions and can be stored at low temperatures, such as -20°C or -80°C, for extended periods to preserve their activity.

Safety consideration in food processing[edit]

When aminopeptidases are used in food processing, it is crucial to ensure that they are food-grade and safe for consumption. Aminopeptidases from A. oryzae and A. sojae, for example, have been extensively studied and are considered safe for use in food applications.[20] It is important to handle these enzymes under conditions that prevent contamination and degradation, which could affect both the safety and quality of the food products.

Compliance with safety regulations[edit]

Compliance with safety regulations is essential when using aminopeptidases in food processing. Regulatory frameworks are in place to ensure that food-grade enzymes meet safety standards. For instance, the review on innovative approaches in food processing highlights the importance of adhering to advanced technologies and regulatory frameworks to ensure food quality, preservation, and safety.[20] Additionally, understanding factors leading to non-compliance with agri-food safety regulations can help improve adherence to these standards, ensuring that food products are safe for consumers.[21]

See also[edit]

References[edit]

  1. ^ a b PDB: 3QNF​: Kochan G, Krojer T, Harvey D, Fischer R, Chen L, Vollmar M, von Delft F, Kavanagh KL, Brown MA, Bowness P, Wordsworth P, Kessler BM, Oppermann U (May 2011). "Crystal structures of the endoplasmic reticulum aminopeptidase-1 (ERAP1) reveal the molecular basis for N-terminal peptide trimming". Proceedings of the National Academy of Sciences of the United States of America. 108 (19): 7745–50. Bibcode:2011PNAS..108.7745K. doi:10.1073/PNAS.1101262108. PMC 3093473. PMID 21508329.
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  21. ^ Bunei, Emmanuel K.; Barclay, Elaine; Kotey, Bernice (2023-09-27). "Understanding Factors Leading to Farmer Non-compliance with Agri-food Safety Regulations in Kenya: A Quantitative Analysis". International Journal of Rural Criminology. 8 (1): 59–81. doi:10.18061/ijrc.v8i1.9564. ISSN 2768-3109.

External links[edit]