| Trypsin and protease inhibitor | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
 Structure of a Kunitz-type trypsin inhibitor. [1] | |||||||||||
| Identifiers | |||||||||||
| Symbol | Kunitz_legume | ||||||||||
| Pfam | PF00197 | ||||||||||
| InterPro | IPR002160 | ||||||||||
| PROSITE | PDOC00255 | ||||||||||
| SCOP2 | 1tie / SCOPe / SUPFAM | ||||||||||
| |||||||||||
Kunitz soybean trypsin inhibitor is a type of protein contained in legume seeds which functions as a protease inhibitor. [2] Kunitz-type Soybean Trypsin Inhibitors are usually specific for either trypsin or chymotrypsin. They are thought to protect seeds against consumption by animal predators.
Two types of trypsin inhibitors are found in soy: the Kunitz-type soybean trypsin inhibitor (STI, discovered by Moses Kunitz and sometimes abbreviated as KTI) and the Bowman-Birk inhibitor (BBI). STI is a large (20,100 daltons), strong inhibitor of trypsin, while BBI is much smaller (8,000 daltons) and inhibits both trypsin and chymotrypsin. [3] Both inhibitors have significant anti-nutritive effects in the body, affecting digestion by hindering protein hydrolysis and activation of other enzymes in the gut. STI is found in much larger concentrations than BBI in soy, however, to achieve the highest nutritional value from soy, both of these inhibitors must be denatured in some way. Whole soybeans have been reported to contain 17–27 mg of trypsin inhibitor per gram.
Protease inhibitory activity is decreased by cooking soybeans, leading to low levels in soy products such as tofu and soy milk. [4]
Proteins from the Kunitz family contain from 170 to 200 amino acid residues and one or two intra-chain disulfide bonds. The best conserved region is found in their N-terminal section. The crystal structures of soybean trypsin inhibitor (STI), trypsin inhibitor DE-3 from the coral tree Erythrina afra (ETI) [1] and the bifunctional proteinase K/alpha-amylase inhibitor from wheat (PK13) have been solved, showing them to share the same beta trefoil fold structure as those of interleukin 1 and heparin-binding growth factors. [5]
Despite the structural similarity, STI shows no interleukin-1 bioactivity, presumably as a result of their primary sequence disparities. The active inhibitory site containing the scissile bond is located in the loop between beta-strands 4 and 5 in STI and ETI.
Trypsin inhibitors require a specific three-dimensional structure in order to inactivate trypsin in the body. They bind strongly to trypsin, blocking its active site and instantly forming a highly stable adduct and halting digestion of certain proteins. Trypsin, a serine protease, is responsible for cleaving the polypeptide backbone following arginine or lysine.
After a meal, trypsinogen release is stimulated by cholecystokinin and undergoes specific proteolysis for activation. Free trypsin is then able to activate other serine proteases, such as chymotrypsin, elastase, and more trypsin (by autocatalysis), or continue breaking down proteins. [6] However, if trypsin inhibitors (specifically STI) are present, the majority of trypsin in the cycle of digestion is inactivated and ingested proteins remain whole. Effects of this occurrence include gastric distress, and pancreatic hyperplasia (proliferation of cells) or hypertrophy (enlargement of cells). [7]
The amount of soy inhibitors is directly related to the amount of trypsin it will inhibit, therefore a product with high concentration of soy is likely to produce large values of inhibition. In a rat model, animals were fed either soy protein concentrate or direct concentrate of STI. In both instances, after a week the rats showed a dose-related increase in pancreas weight due to both hyperplasia and hypertrophy. [7] This indicates that long-term consumption of a diet high in soy with strong trypsin inhibitor activity may produce unwanted effects in humans as well.
A significant amount of research is being done to determine the best method of inhibitor inactivation. The most successful methods found so far include:
STI is highly resistant to pepsin, enabling STI to avoid degradation in the stomach and then inhibit trypsin. Hence STI was engineered into an antibody mimetic called a gastrobody, aiming to address the problems of antibody degradation in the gut following oral delivery. Loops of STI were randomized and selected by phage display for binding to a target of interest (a toxin from Clostridioides difficile). [8]
While trypsin inhibitors have been widely regarded as anti-nutritive factors in soy, research is currently being done on the inhibitors’ possible anti-carcinogenic characteristics. Some research has shown that protease inhibitors can cause irreversible suppressive effect on carcinogenic cell growth. However, the mechanism is still unknown. The cancers showing positive results for this new development are colon, oral, lung, liver, and esophageal cancers. Further research is still necessary to determine things such as the method of delivery for this natural anti-carcinogen, as well as performing extensive clinical trials in this area. [9]
Proteolysis is the breakdown of proteins into smaller polypeptides or amino acids. Uncatalysed, the hydrolysis of peptide bonds is extremely slow, taking hundreds of years. Proteolysis is typically catalysed by cellular enzymes called proteases, but may also occur by intra-molecular digestion.
Trypsin is an enzyme in the first section of the small intestine that starts the digestion of protein molecules by cutting long chains of amino acids into smaller pieces. It is a serine protease from the PA clan superfamily, found in the digestive system of many vertebrates, where it hydrolyzes proteins. Trypsin is formed in the small intestine when its proenzyme form, the trypsinogen produced by the pancreas, is activated. Trypsin cuts peptide chains mainly at the carboxyl side of the amino acids lysine or arginine. It is used for numerous biotechnological processes. The process is commonly referred to as trypsinogen proteolysis or trypsinization, and proteins that have been digested/treated with trypsin are said to have been trypsinized.
A protease is an enzyme that catalyzes proteolysis, breaking down proteins into smaller polypeptides or single amino acids, and spurring the formation of new protein products. They do this by cleaving the peptide bonds within proteins by hydrolysis, a reaction where water breaks bonds. Proteases are involved in numerous biological pathways, including digestion of ingested proteins, protein catabolism, and cell signaling.
Pepsin is an endopeptidase that breaks down proteins into smaller peptides and amino acids. It is one of the main digestive enzymes in the digestive systems of humans and many other animals, where it helps digest the proteins in food. Pepsin is an aspartic protease, using a catalytic aspartate in its active site.
In biology and biochemistry, protease inhibitors, or antiproteases, are molecules that inhibit the function of proteases. Many naturally occurring protease inhibitors are proteins.
Serine proteases are enzymes that cleave peptide bonds in proteins. Serine serves as the nucleophilic amino acid at the (enzyme's) active site. They are found ubiquitously in both eukaryotes and prokaryotes. Serine proteases fall into two broad categories based on their structure: chymotrypsin-like (trypsin-like) or subtilisin-like.
Digestive enzymes take part in the chemical process of digestion, which follows the mechanical process of digestion. Food consists of macromolecules of proteins, carbohydrates, and fats that need to be broken down chemically by digestive enzymes in the mouth, stomach, pancreas, and duodenum, before being able to be absorbed into the bloodstream. Initial breakdown is achieved by chewing (mastication) and the use of digestive enzymes of saliva. Once in the stomach further mechanical churning takes place mixing the food with secreted gastric acid. Digestive gastric enzymes take part in some of the chemical process needed for absorption. Most of the enzymatic activity, and hence absorption takes place in the duodenum.
Enteropeptidase is an enzyme produced by cells of the duodenum and is involved in digestion in humans and other animals. Enteropeptidase converts trypsinogen into its active form trypsin, resulting in the subsequent activation of pancreatic digestive enzymes. Absence of enteropeptidase results in intestinal digestion impairment.
A catalytic triad is a set of three coordinated amino acids that can be found in the active site of some enzymes. Catalytic triads are most commonly found in hydrolase and transferase enzymes. An acid-base-nucleophile triad is a common motif for generating a nucleophilic residue for covalent catalysis. The residues form a charge-relay network to polarise and activate the nucleophile, which attacks the substrate, forming a covalent intermediate which is then hydrolysed to release the product and regenerate free enzyme. The nucleophile is most commonly a serine or cysteine amino acid, but occasionally threonine or even selenocysteine. The 3D structure of the enzyme brings together the triad residues in a precise orientation, even though they may be far apart in the sequence.
A trypsin inhibitor (TI) is a protein and a type of serine protease inhibitor (serpin) that reduces the biological activity of trypsin by controlling the activation and catalytic reactions of proteins. Trypsin is an enzyme involved in the breakdown of many different proteins, primarily as part of digestion in humans and other animals such as monogastrics and young ruminants. Serpins – including trypsin inhibitors – are irreversible and suicide substrate-like inhibitors.
Trypsin-1, also known as cationic trypsinogen, is a protein that in humans is encoded by the PRSS1 gene. Trypsin-1 is the main isoform of trypsinogen secreted by pancreas, the others are trypsin-2, and trypsin-3 (meso-trypsinogen).
Suppressor of tumorigenicity 14 protein, also known as matriptase, is a protein that in humans is encoded by the ST14 gene. ST14 orthologs have been identified in most mammals for which complete genome data are available.
Kunitz-type protease inhibitor 2 is an enzyme inhibitor that in humans is encoded by the SPINT2 gene. SPINT2 is a transmembrane protein with two extracellular Kunitz domains to inhibit serine proteases. This gene is a presumed tumor suppressor by inhibiting HGF activator which prevents the formation of active hepatocyte growth factor. Mutations in SPINT2 could result in congenital sodium diarrhea (CSD).
Serpin B6 is a protein that in humans is encoded by the SERPINB6 gene.
Kunitz domains are the active domains of proteins that inhibit the function of protein degrading enzymes or, more specifically, domains of Kunitz-type are protease inhibitors. They are relatively small with a length of about 50 to 60 amino acids and a molecular weight of 6 kDa. Examples of Kunitz-type protease inhibitors are aprotinin, Alzheimer's amyloid precursor protein (APP), and tissue factor pathway inhibitor (TFPI). Kunitz STI protease inhibitor, the trypsin inhibitor initially studied by Moses Kunitz, was extracted from soybeans.
In molecular biology the protein SSI is a Subtilisin inhibitor-like which stands for Streptomyces subtilisin inhibitor. This is a protease inhibitor. These are often synthesised as part of a larger precursor protein, either as a prepropeptide. The function of this protein domain is to prevent access of the substrate to the active site. It is found only in bacteria.
In molecular biology, the Bowman–Birk protease inhibitor family of proteins consists of eukaryotic proteinase inhibitors, belonging to MEROPS inhibitor family I12, clan IF. They mainly inhibit serine peptidases of the S1 family, but also inhibit S3 peptidases.
The Kazal domain is an evolutionary conserved protein domain usually indicative of serine protease inhibitors. However, kazal-like domains are also seen in the extracellular part of agrins, which are not known to be protease inhibitors.
In molecular biology the β trefoil fold is a protein fold that consists of six beta hairpins, each formed by two beta strands. Together these form a beta barrel with a triangular "cap", each consisting of three hairpins. Overall, this structure has an approximate three-fold symmetry.
Moses Kunitz was a Russian-American biochemist who spent most of his career at Rockefeller University. He is best known for a series of experiments in purification and crystallization of proteins, contributing to the determination that enzymes are proteins.