Trypsin and Chymotrypsin
By Jennifer McDowall
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trypsin/chymotrypsin structure
Digestive enzymes, synthesized and secreted by pancreatic acinar cells, breakdown the foods that we eat: starches (amylases), fats (lipases) and proteins (proteases). Pancreatic proteases have long been used in medicine both diagnostically and therapeutically for pancreatic disease, and more recently for their involvement in cancer metastases. Proteases represent a diverse array of enzymes that act on the peptide bonds within proteins. They can be divided into discreet protein families that differ with respect to structure and catalytic type, including serine, threonine, cysteine, aspartic and metallo proteases. The two proteases trypsin and chymotrypsin are grouped into the serine protease family.
Trypsin and chymotrypsin, serine protease
digestive enzymes.
Trypsin and chymotrypsin, like most proleotytic enzymes, are synthesized as inactive zymogen precursors (trypsinogen and chymotrypsinogen) to prevent unwanted destruction of cellular proteins, and to regulate when and where enzyme activity occurs. The inactive zymogens are secreted into the duodenum, where they travel the small and large intestines prior to excretion. Zymogens also enter the bloodstream, where they can be detected in serum prior to excretion in urine. Zymogens are converted to the mature, active enzyme by proteolysis to split off a pro-peptide, either in a subcellular compartment or in an extracellular space where they are required for digestion.
Trypsin and chymotrypsin are structurally very similar, although they recognise different substrates. Trypsin acts on lysine and arginine residues, while chymotrypsin acts on large hydrophobic residues such as tryptophan, tyrosine and phenylalanine, both with extraordinary catalytic efficiency. Both enzymes have a catalytic triad of serine, histidine and aspartate within the S1 binding pocket, although the hydrophobic nature of this pocket varies between the two, as do other structural interactions beyond the S1 pocket.
The human pancreas secretes three isoforms of trypsinogen: cationic (trypsinogen-1), anionic (trypsinogen-2) and mesotrypsinogen (trypsinogen-3). Cationic and anionic trypsins are the major isoforms responsible for digestive protein degradation, occurring in a ratio of 2:1, while mesotrypsinogen accounts for less than 5% of pancreatic secretions. Mesotrypsin is a specialised protease known for its resistance to trypsin inhibitors. It is thought to play a special role in the degradation of trypsin inhibitors, possibly to aid in the digestion of inhibitor-rich foods such as soybeans and lima beans. An alternatively spliced mesotrypsinogen in which the signal peptide is replaced with a different exon 1 is expressed in the human brain; the function of this brain trypsinogen is unknown.
There are two isoforms of pancreatic chymotrypsin, A and B, which are known to cleave proteins selectively at specific peptide bonds formed by the hydrophobic residues tryptophan, phenylalanine and tyrosine.
The need for inhibitors
Proteases perform many beneficial functions that are essential to life, but uncontrolled they can be dangerous. Protease inhibitors are used as the major form of control once the protease has been activated. In higher organisms, there is a delicate balance between proteases and their natural inhibitors to help control the activation and catabolism of many intra- and extra-cellular proteins. In mammals, the bloodstream is a major carrier for many glycoproteins that act as protease inhibitors. Protease inhibitors use a reactive site peptide bond to serve as a substrate for various proteases, forming very stable complexes where the inhibitor peptide bond is hydrolysed by the protease extremely slowly, thereby effectively removing the protease from circulation. There are at least eighteen families of protease inhibitors, all of which share a common conformation surrounding the reactive site peptide bond, even though they differ in their global structure. Some members only hydrolyse trypsin, such as chicken ovomucoid, while others hydrolyse both trypsin and chymotrypsin using different inhibitory domains, such as turkey ovomucoid.
Protease inhibitors are ubiquitous in nature. They are widely distributed in plant seeds, particularly in legumes. The presence of these inhibitors in seeds acts as a feeding deterrent, especially in insects where they inhibit midgut proteases. Inhibitors can deter other animals as well; isolated soybean inhibitors have been found to cause enlargement of the pancreas in certain species, such as rat and mouse. Many bacterial species produce protease inhibitors that help them to survive the digestive processes of the gut, such as ecotin in Escherichia coli, which is effective against several different pancreatic proteases because of its flexibility.
Protease inhibitors can have nutritional value as well. The Bowman-Birk inhibitor (BBI) from soybean may play a role in the prevention of tumourigenesis. BBI is also an effective inhibitor of nephrotoxicity induced by the antibiotic gentamicin.