Structural Biochemistry/Enzyme Regulation/Proteolytic activation
Proteolytic Activation is the activation of an enzyme by peptide cleavage. The enzyme is initially transcribed in a longer, inactive form. In this enzyme regulation process, the enzyme is shifted between the inactive and active state. Irreversible conversions can occur on inactive enzymes to become active. This inactive precursor is known as a zymogen or a proenzyme. The enzyme is subsequently cut to yield the active form. The benefit of this type of regulation is that ATP is not needed for the cleavage to occur. Therefore, enzymes can use this system of regulation even outside of the cell. This type of regulation can only be done once to an enzyme. It can only be activated once and will stay activated for the enzyme's entire life span. Unlike allosteric control and reversible covalent modification this occurs just once in an enzyme's lifetime. Although the zymogen activation is irreversible, there are specific inhibitors that control those proteases.
Specific proteolysis is a frequent way of activating enzymes and certain other proteins in biological systems. For instance, digestive enzymes which hydrolyze proteins are made as zymogens in the stomach and pancreas, in which pepsinogen is the inactive precursor (zymogen) and pepsin is the activated form of the enzyme. Another example is seen in blood clotting which is carried out by a cascade of proteolytic activations. Also, certain protein hormones are synthesizes as the inactive precursors such as proinsulin which then leads to the activated form insulin by proteolytic cleavage. In addition, Collagen, a fibrous protein and the major component of skin and bone is made from the zymogen procollagen.
Several developmental process are mediated by activation of inactive precursors such as the metamorphosis of a tadpole to a full grown frog in which increases amounts of collage are reabsorbed from the tail. Similarly, collage is decomposed in mammaliam uterus after birth. Both of these examples rely on the conversion of procollagenase to collagenase which is the active protease and is very accurately timed.
Lastly, apoptosis, or programmed cell death is another example that illustrates the importance of proteolytic enzymes, in this case caspases. Caspases are synthesized in the inactive form called procaspases and when activated by different signals, caspases cause cell death in many organisms.
Common uses in Biological Systems
Digestive enzymes are activated in the stomach and pancreas using this methodology. They are synthesized first as zymogens. Pepsinogen is the inactive protein which is initially transcribed. Then, it is cleaved in the stomach to produce Pepsin, a digestion enzyme. The purpose of this regulation is to prevent the Pepsin from digesting proteins in the body before it is introduced into the digestive tract. Many protein hormones in the body originate from inactivated forms of the actual enzymes. A prime example is insulin. Insulin arises from an inactive form known as proinsulin. It is activated by proteolytic cleavage of a specific peptide. Proinsulin is first synthesized in the endoplasmic reticulum where the peptide chain is folded and the disulfide bonds oxidized. It is then packaged in the Golgi Apparatus and it is also proteolyically cleaved by series of proteases to form insulin. The matured insulin has 39 less amino acids than the proinsulin: 4 are removed and recycled and the remaining 35 amino acids form the C-peptide. Collagen is a fibrous protein that makes up the majority of the components of connective tissues in animals that arises from a inactive form known as procollagen. Also many developments and processes are activated by proenzymes. Programmed cell death is also mediated by these types of proteolytic enzymes.
An enzyme that hydrolyzes proteins, cleaving the peptide bond. The inactive form of this enzyme is chymotrypsinogen. The zymogen is synthesized in the pancreas, where most of the secreting proteins are synthesized. Chymotrypsinogen consist of the 245 amino acids. In order to activate it, the bond in between the 15th amino acid (Arg) and 16th amino acid (Ile) has to be cleaved by the enzyme (trypsin). The simple cleavage of the single bond activates the enzyme. Later on, the two dipeptides can be removed to produce α-chymotrypsin. Three chains in α-chymotrypsin are bonded by disulfide bonds. The cleavage of a single bond leads to the structure conformation and creation of the active side of the chymotrypsin (catalytic triad). It also creates a pocket where the aromatic or long hydrophilic side chain of the amino acid can be inserted for the future cleavage. In addition, structural change locates the NH group that stabilized the tetrahedral intermediate in the “oxyanion hole” in the appropriate position.
As a proteoplytic activation, chymotrypsin has been biologically known to work as digestive enzymes, blood clotting, protein hormones, and procaspases (a programmed cell death).
Important enzyme in the biological systems because it activates many enzymes. However, trypsin by itself is activated from the trypsinogen with help of the enteropeptidase. Enteropeptidase is a serine protease just like trypsin and chymotrypsin, and it breaks Lys-Ile bond in the trypsinogen and activates it. As a result of such process, the small amount of the trypsin enzyme is produced. Later, this amount of activated trypsin activates more trypsin and other enzymes too. The formation of trypsin by enteropeptidase is considered the master activaion step since trypsin participates in a variety of zymogen activation. Examples include the activation of proelastase to elastaase, procarboxypeptidase to carboxypeptidase, and prolipase to lipase.
An important role of zymogen activations occurs in blood clotting. For blood clotting, the response time must be fast in order to achieve clotting at the right spot and time to prevent excessive bleeding. Enzymatic cascades are therefore employed to achieve that rapid response. A cascade of zymogen activations activates a clotting factor, which is then responsible for activating another clotting factor and so forth until the final clot is achieved. The blood clotting process is driven by a series of proteolytic events. When trauma exposes tissue factor, thrombin, also a serine protease and a key enzyme in clotting, is synthesized. This event leads to the production of more thrombin by positive feedback. Thrombin then activates enzymes and factors such as fibrinogen and forms fibrin, the key part in blood clotting. Thrombin cleaves four arginine-glycine peptide bonds on the in the central globular region of fibrinogen, releasing fibrinopeptides. The fibrinogen molecule that has lost these fibrinopeptides are then called fibrin monomers. They are called monomers because they spontaneously come together and assemble into fibrous arrays known as fibrin. The fibrins are then crosslinked by the enzyme transglutaminase, which was activated by thrombin from protransglutaminase.
Breakthroughs in Elucidation of Clotting Pathways
Because of the breakthrough in the elucidation of blood clotting pathways, hemophilia can be revealed early in clotting. Classic hemophilia (a.k.a. Hemophilia A) is a clotting defect. It is genetically transmitted as a sex-linked recessive characteristic. The antihemophilic factor, factor VIII of the pathway is missing or has reduced activity. Even though factor VIII is not a protease, it stimulates the activation of factor X which is the final protease of the intrinsic pathway by the serine protease factor IXa. The activity of factor VIII is increased by limited proteolysis by thrombin. This type of positive feedback amplifies the clotting singal and accelerates clot formation after a threshold has been reached. Therefore the activation of the intrinsic pathway is impaired in classic hemophilia.
Before, hemophiliacs were treated with transfusions of concentrated plasma fraction with factor VIII but this therapy always had the risk of infection of diseases such as hepatitis and AIDS. However, with advancements in biochemical techniques such as biochemical purification and recombinant DNA, the gene that encodes factor VIII was isolated and expressed in cell cultures. Since then recombinant factor VIII purified from the cultures has replaced plasma concentrates to treat hemophilia.
Vitamin K-Dependent Modification readies the activation of prothrombin
Thrombin is produced as a zymogen known as prothrombin. The inactive molecule has four domains. The first domain is the gla domain ( a gamma-carboxyglutamate-rich domain). Kringle domains consist of the next two domains. Kringle domains keep prothrombin in an inactive form and guide it towards appropriate site for activation by factor Xa (serine protease) and factor Va (stimulatory protein). The activation begins with proteolytic cleavage of the arginine 274 and threonine 275 bond, which releases a fragment containing the first three domains. Similar cleavage of the arginine 323 and isoleucine 324 bond generates an active thrombin molecule.
Vitamin K has an important role in the synthesis of prothrombin. Nuclear magnetic resonance reveals that prothrombin contains gamma-carboxyglutamate. The first 10 glutamate residues in the amino-terminal region of prothrombin are carboxylated to gamma-carboxyglutamate by a vitamin K-dependent enzyme. This reaction converts glutamate, a weak chelator of Ca 2+, into gamma-carboxyglutamate, a strong chelator. Consequently, prothrombin is able to bind calcium. This binding fixes the zymogen to the phospholipid membrane surface from blood platelets at the injury site. This is important because the prothrombin is now in close proximity to two clotting proteins that catalyze its conversion to thrombin. The calcium-binding domain is removed during activation, which frees the thrombin from the membrane. Now, it is free to cleave targets such as fibrinogen.
Prothrombin synthesized in the absence of vitamin K or in the presene of vitamin K antagonists such as dicourmaorl gives rise to an anticoagulant factor as opposed to the regular thrombin that is a coagulant factor. An anticoagulant factor prevents the blood from clotting and is used to treat patients with thrombosis.
The abnormal prothrombin lacks the γ-Carboxyglutamate and that explains its anticoagulant properties.
The 26S proteasome is an enzyme that is known to cleave intracellular proteins in order to maintain cell functioning and homeostasis. One of the pathways that the 26S proteasome is known for is the degradation of ubiquitin. This proteasome is composed of over 30 subunits and all of their units have specific functions that are not limited to unfolding, translocating, and cleaving. As the 26S proteasome recognizes the correct ubiquitin, it forms the UPS-ubiquitin-proteasome system. The UPS is now polyubiquinated and Ubiquitin chains are formed. The chains are now available to be picked out by the proteolytic core of the UPS, which will continue on with the cleaving of ubiquitin into small peptides. This process is more complicated than it appears, even with the structure of the 26S proteasome not fully understood. It is composed of two subunits: the 20S proteasome core particle and the 19S regulatory particle. The proteasome core particle is where substrate proteolysis occurs and then regulatory particle is where selecting and unfolding of ubiquitin substrates occurs. The core particle has a barrel type of structure with many alpha and beta subunits arranged in alternating fashion. Such a complex structure also defines its function because it requires great amounts of cellular energy to assemble and degrade the molecule of interest. The regulatory particle is just as complex, for it contains of a base and a lid. The base is made up of six different AAA ATPases and by three non ATPase subunits. This helps the regulatory particle control the entry of substrates into the previously mentioned core particle, and hence control the overall pace of the assembly and rate of the substrate-enzyme complex mechanism.
Source: The 26S proteasome: assembly and function of a destructive machine Center for Integrated Protein Science at the Department Chemie, Lehrstuhl für Biochemie, Technische Universität München, Lichtenbergstr. 4, 85747 Garching, Germany
Proteolytic Enzyme Inhibitors
Since activating proteolytic enzymes is irreversible, how can one prevent a proteolytic enzyme from functioning? These types of enzymes need very specific inhibitors to prevent the enzyme from running catalysis. A prime example is pancreatic trypsin inhibitor. This inhibits trypsin by binding really firmly to the active site of the enzyme. What makes it bind firmly to the active site is interactions with the aspartate side chain with the side chain of lysine 15 on the inhibitor. This along with many hydrogen bonds between the inhibitor and the active site creates a relatively firm binding on the active site.
Trypsin inhibitors are essential in that they prevent severe damage such as inflammation in the pancreas known as pancreatitis. Because trypsin engages in activation of several zymogens, inhibiting trypsin can prevent unwanted or premature cascades such as inflammation. Other inhibitors, such as antielastase, are important as well. When the body contains excess elastase, the enzyme destroys alveolar walls in the lungs by breaking down elastic fibers and other connective tissue proteins in the lungs. This disease is called emphysema, containing symptoms such as difficulty in breathing. Therefore, the body needs elastase inhibitors to prevent the damage from occurring.
When the body forms blood clots, there also needs to be regulation that makes sure the clot is limited only to the site of injury. An example includes thrombin, which not only catalyzes the formation of the clot through fibrin, but also deactivates the clotting cascade by activating protein C, which digests clot stimulating factors. Other specific clotting inhibitors include antithrombin III, which forms an irreversible complex with thrombin, and thus preventing thrombin from activating any more fibrinogen. Antithrombin III can also be amplified by heparin, a polysaccharide found in the walls of blood vessels and endothelial cells. Heparin amplifies antithrombin III by increasing the rate of formation of the irreversible antithrombin III-thrombin complex.
The ratio of thrombin to antithrombin is crucial in normal blood clotting. A mutation that occurs in α1-antitrypsin , a protease inhibitor, which replaces the methionine 358 with arginine, alter the inhibitor's specificity from elastase to thrombin and causes the level of thrombin to drop at the injury site. neutrophils produce large quantities of elastase at sites of injury and those excess proteases must be inhibited by α1-antitrypsin, however, the mutant α1-antitrypsin will inhibit the coagulation factor thrombin instead of elstase thus the blood clot will fail to form and the patient can die of potential hemorrhage.
Berg, Jeremy M. John L. Tymoczko. Lubert Stryer. Biochemistry Sixth Edition. New York: W.H. Freeman, and Company 2007.