We turn now to a different mechanism of enzyme regulation. Many enzymes acquire full enzymatic activity as they spontaneously fold into their characteristic three-dimensional forms. In contrast, the folded forms of other enzymes are inactive until the cleavage of one or a few specific peptide bonds. The inactive precursor is called a zymogen or a proenzyme. An energy source such as ATP is not needed for cleavage. Therefore, in contrast with reversible regulation by phosphorylation, even proteins located outside cells can be activated by this means. Another noteworthy difference is that proteolytic activation, in contrast with allosteric control and reversible covalent modification, is irreversible, taking place just once in the life of an enzyme molecule.

Specific proteolysis is a common means of activating enzymes and other proteins in biological systems. For example:

We next examine the activation and control of zymogens, using enzymes responsible for digestion and blood-clot formation as examples.

Chymotrypsin is a digestive enzyme that hydrolyzes proteins. Chymotrypsin, whose mechanism of action was described in detail in Chapter 9, specifically cleaves peptide bonds on the carboxyl side of amino acid residues with large, hydrophobic R groups (Table 8.6). Its inactive precursor, chymotrypsinogen, is synthesized in the pancreas, as are several other zymogens and digestive enzymes. Indeed, the pancreas is one of the most active organs in synthesizing and secreting proteins. The enzymes and zymogens are synthesized in the acinar cells of the pancreas and stored inside membrane-bounded granules (Figure 10.19). The zymogen granules accumulate at the apex of the acinar cell; when the cell is stimulated by a hormonal signal or a nerve impulse, the contents of the granules are released into a duct leading into the duodenum.

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Chymotrypsinogen, a single polypeptide chain consisting of 245 amino acid residues, is virtually devoid of enzymatic activity. It is converted into a fully active enzyme when the peptide bond joining arginine 15 and isoleucine 16 is cleaved by trypsin (Figure 10.20). The resulting active enzyme, called π-chymotrypsin, then acts on other π-chymotrypsin molecules by removing two dipeptides to yield α-chymotrypsin, the stable form of the enzyme. The three resulting chains in α-chymotrypsin remain linked to one another by two interchain disulfide bonds. The striking feature of this activation process is that cleavage of a single specific peptide bond transforms the protein from a catalytically inactive form into one that is fully active.

How does cleavage of a single peptide bond activate the zymogen? The cleavage of the peptide bond between amino acids 15 and 16 triggers key conformational changes, which were revealed by the elucidation of the three-dimensional structure of chymotrypsinogen.

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The structural changes accompanying the activation of trypsinogen, the precursor of the proteolytic enzyme trypsin, are different from those in the activation of chymotrypsinogen. Four regions of the polypeptide are very flexible in the zymogen, whereas they have a well-defined conformation in trypsin. The resulting structural changes also complete the formation of the oxyanion hole.

The digestion of proteins and other molecules in the duodenum requires the concurrent action of several enzymes, because each is specific for a limited number of side chains. Thus, the zymogens must be switched on at the same time. Coordinated control is achieved by the action of trypsin as the common activator of all the pancreatic zymogens —trypsinogen, chymotrypsinogen, proelastase, procarboxypeptidase, and prolipase, the inactive precursor of a lipid-degrading enzyme. To produce active trypsin, the cells that line the duodenum display a membrane-embedded enzyme, enteropeptidase, which hydrolyzes a unique lysine–isoleucine peptide bond in trypsinogen as the zymogen enters the duodenum from the pancreas. The small amount of trypsin produced in this way activates more trypsinogen and the other zymogens (Figure 10.22). Thus, the formation of trypsin by enteropeptidase is the master activation step.

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The conversion of a zymogen into a protease by cleavage of a single peptide bond is a precise means of switching on enzymatic activity. However, this activation step is irreversible, and so a different mechanism is needed to terminate proteolysis. Specific protease inhibitors accomplish this task. Serpins, serine protease inhibitors, are an example of one such family of inhibitors. For example, pancreatic trypsin inhibitor, a 6-kDa protein, inhibits trypsin by binding very tightly to its active site. The dissociation constant of the complex is 0.1 pM, which corresponds to a standard free energy of binding of about −75 kJ mol⁻¹ (− 18 kcal mol⁻¹). In contrast with nearly all known protein assemblies, this complex is not dissociated into its constituent chains by treatment with denaturing agents such as 8 M urea or 6 M guanidine hydrochloride.

The reason for the exceptional stability of the complex is that pancreatic trypsin inhibitor is a very effective substrate analog. X-ray analyses show that the inhibitor lies in the active site of the enzyme, positioned such that the side chain of lysine 15 of this inhibitor interacts with the aspartate side chain in the specificity pocket of trypsin. In addition, there are many hydrogen bonds between the main chain of trypsin and that of its inhibitor. Furthermore, the carbonyl group of lysine 15 and the surrounding atoms of the inhibitor fit snugly in the active site of the enzyme. Comparison of the structure of the inhibitor bound to the enzyme with that of the free inhibitor reveals that the structure is essentially unchanged on binding to the enzyme (Figure 10.23). Thus, the inhibitor is preorganized into a structure that is highly complementary to the enzyme’s active site. Indeed, the peptide bond between lysine 15 and alanine 16 in pancreatic trypsin inhibitor is cleaved but at a very slow rate: the half-life of the trypsin–inhibitor complex is several months. In essence, the inhibitor is a substrate, but its intrinsic structure is so nicely complementary to the enzyme’s active site that it binds very tightly, rarely progressing to the transition state and is turned over slowly.

The amount of trypsin is much greater than the amount of inhibitor. Why does trypsin inhibitor exist? Recall that trypsin activates other zymogens. Consequently, the prevention of even small amounts of trypsin from initiating the cascade while the zymogens are still in the pancreas or pancreatic ducts is vital. Trypsin inhibitor binds to any prematurely activated trypsin molecules in the pancreas or pancreatic ducts. This inhibition prevents severe damage to those tissues, which could lead to acute pancreatitis.

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Pancreatic trypsin inhibitor is not the only important protease inhibitor. A 53-kDa plasma protein, α₁-antitrypsin (also called α₁-antiproteinase), protects tissues from digestion by elastase, a secretory product of neutrophils (white blood cells that engulf bacteria). Antielastase would be a more accurate name for this inhibitor, because it blocks elastase much more effectively than it blocks trypsin. Like pancreatic trypsin inhibitor, α₁-antitrypsin blocks the action of target enzymes by binding nearly irreversibly to their active sites. Genetic disorders leading to a deficiency of α₁-antitrypsin illustrate the physiological importance of this inhibitor. For example, the substitution of lysine for glutamate at residue 53 in the type Z mutant slows the secretion of this inhibitor from liver cells. Serum levels of the inhibitor are about 15% of normal in people homozygous for this defect. The consequence is that excess elastase destroys alveolar walls in the lungs by digesting elastic fibers and other connective-tissue proteins.

The resulting clinical condition is called emphysema (also known as chronic obstructive pulmonary disease [COPD]). People with emphysema must breathe much harder than normal people to exchange the same volume of air because their alveoli are much less resilient than normal. Cigarette smoking markedly increases the likelihood that even a type Z heterozygote will develop emphysema. The reason is that smoke oxidizes methionine 358 of the inhibitor (Figure 10.24), a residue essential for binding elastase. Indeed, this methionine side chain is the bait that selectively traps elastase. The methionine sulfoxide oxidation product, in contrast, does not lure elastase, a striking consequence of the insertion of just one oxygen atom into a protein and a remarkable example of the effect of human behavior on biochemistry. We will consider another protease inhibitor, antithrombin III, when we examine the control of blood clotting.

Enzymatic cascades are often employed in biochemical systems to achieve a rapid response. In a cascade, an initial signal institutes a series of steps, each of which is catalyzed by an enzyme. At each step, the signal is amplified. For instance, if a signal molecule activates an enzyme that in turn activates 10 enzymes and each of the 10 enzymes in turn activates 10 additional enzymes, after four steps the original signal will have been amplified 10,000-fold. Hemostasis, the process of blood clot formation and dissolution, requires a cascade of zymogen activations: the activated form of one clotting factor catalyzes the activation of the next (Figure 10.25). Thus, very small amounts of the initial factors suffice to trigger the cascade, ensuring a rapid response to trauma.

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Two means of initiating blood clotting have been described, the intrinsic pathway and the extrinsic pathway. The intrinsic clotting pathway is activated by exposure of anionic surfaces upon rupture of the endothelial lining of the blood vessels. The extrinsic pathway, which appears to be most crucial in blood clotting, is initiated when trauma exposes tissue factor (TF), an integral membrane glycoprotein. Upon exposure to the blood, tissue factor binds to factor VII to activate factor X. Both the intrinsic and extrinsic pathways lead to the activation of factor X (a serine protease), which in turn converts prothrombin into thrombin, the key protease in clotting. Thrombin then amplifies the clotting process by activating enzymes and factors that lead to the generation of yet more thrombin, an example of positive feedback. Note that the active forms of the clotting factors are designated with a subscript “a,” whereas factors that are activated by thrombin are designated with an asterisk.

Thrombin is synthesized as a zymogen called prothrombin. The inactive molecule comprises four major domains, with the serine protease domain at its carboxyl terminus (Figure 10.26). The first domain, called the gla domain, is rich in γ carboxyglutamate residues (abbreviation gla), and the second and third domains are called kringle domains (named after a Danish pastry that they resemble). Vitamin K is required for the synthesis of γ carboxyglutamate, a strong chelator of Ca²⁺. These three domains work in concert to keep prothrombin in an inactive form. Moreover, because it is rich in γ carboxyglutamate, the gla domain is able to bind Ca²⁺ (Figure 10.27). What is the effect of this binding? The binding of Ca²⁺ by prothrombin anchors the zymogen to phospholipid membranes derived from blood platelets after injury. This binding is crucial because it brings prothrombin into close proximity to two clotting proteins, factor X_a and factor V_a (a stimulatory protein), that catalyze its conversion into thrombin. Activation is begun by proteolytic cleavage of the bond between arginine 274 and threonine 275 to release a fragment containing the first three domains. Cleavage of the bond between arginine 323 and isoleucine 324 yields active thrombin.

The best-characterized part of the clotting process is the final step in the cascade: the conversion of fibrinogen into fibrin by thrombin. Fibrinogen is made up of three globular units connected by two rods (Figure 10.28). This 340-kDa protein consists of six chains: two each of Aα, Bβ, and γ. The rod regions are triple-stranded α-helical coiled coils, a recurring motif in proteins (Section 2.3). Thrombin cleaves four arginine–glycine peptide bonds in the central globular region of fibrinogen. On cleavage, an A peptide of 18 residues is released from each of the two Aα chains, as is a B peptide of 20 residues from each of the two Bβ chains. These A and B peptides are called fibrinopeptides. A fibrinogen molecule devoid of these fibrinopeptides is called a fibrin monomer and has the subunit structure (αβγ)₂.

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Fibrin monomers spontaneously assemble into ordered fibrous arrays called fibrin. Electron micrographs and low-angle x-ray patterns show that fibrin has a periodic structure that repeats every 23 nm (Figure 10.29). Higher-resolution images reveal how the removal of the fibrinopeptides permits the fibrin monomers to come together to form fibrin. The homologous β and γ chains have globular domains at the carboxyl-terminal ends (Figure 10.30). These domains have binding “holes” that interact with peptides. The β domain is specific for sequences of the form H₃N⁺-Gly-His-Arg-, whereas the γ domain binds H₃N⁺-Gly-Pro-Arg-. Exactly these sequences (sometimes called “knobs”) are exposed at the amino-terminal ends of the β and α chains, respectively, on thrombin cleavage. The knobs of the α subunits fit into the holes on the γ subunits of another monomer to form a protofibril. This protofibril is extended when the knobs of the β subunits fit into the holes of β subunits of other protofibrils. Thus, analogous to the activation of chymotrypsinogen, peptide-bond cleavage exposes new amino termini that can participate in specific interactions. The newly formed “soft clot” is stabilized by the formation of amide bonds between the side chains of lysine and glutamine residues in different monomers.

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This cross-linking reaction is catalyzed by transglutaminase (factor XIII_a), which itself is activated from the protransglutaminase form by thrombin.

Vitamin K (Figure 10.31) has been known for many years to be essential for the synthesis of prothrombin and several other clotting factors. Indeed, it is called vitamin K because a deficiency in this vitamin results in defective blood koagulation (Scandinavian spelling). After ingestion, vitamin K is reduced to a dihydro derivative that is required by γ-glutamyl carboxylase to convert the first 10 glutamate residues in the amino-terminal region of prothrombin into γ-carboxyglutamate (Figure 10.32).

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Recall that γcarboxyglutamate, a strong chelator of Ca²⁺, is required for the activation of prothrombin. Dicoumarol, which is found in spoiled sweet clover, causes a fatal hemorrhagic disease in cattle fed on this hay. Cows fed dicoumarol synthesize an abnormal prothrombin that does not bind Ca²⁺, in contrast with normal prothrombin. Dicoumarol was the first anticoagulant used to prevent thromboses in patients prone to clot formation. However, it is seldom used now because of poor absorption and gastrointestinal side effects. Warfarin, another vitamin K antagonist, is commonly administered as an anticoagulant. Warfarin inhibits the keto reductase and quinone reductase that are required to regenerate the dihydro derivative of vitamin K (Figure 10.32). Dicoumarol, warfarin, and their chemical derivatives serve as effective rat poisons.

There is a fine line between hemorrhage and thrombosis, the formation of blood clots in blood vessels. Clots must form rapidly yet remain confined to the area of injury. What are the mechanisms that normally limit clot formation to the site of injury? The lability of clotting factors contributes significantly to the control of clotting. Activated factors are short-lived because they are diluted by blood flow, removed by the liver, and degraded by proteases. For example, the stimulatory protein factors V_a and VIII_a are digested by protein C, a protease that is switched on by the action of thrombin. Thus, thrombin has a dual function: it catalyzes the formation of fibrin and it initiates the deactivation of the clotting cascade.

Specific inhibitors of clotting factors are also critical in the termination of clotting. For instance, tissue factor pathway inhibitor (TFPI) inhibits the complex of TF–VII_a–X_a that activates thrombin. Another key inhibitor is antithrombin III, a member of the serpin family of protease inhibitors that forms an irreversible inhibitory complex with thrombin. Antithrombin III resembles α₁-antitrypsin except that it inhibits thrombin much more strongly than it inhibits elastase (Figure 10.23). Antithrombin III also blocks other serine proteases in the clotting cascade—namely, factors XII_a, XI_a, IX_a, and X_a. The inhibitory action of antithrombin III is enhanced by heparin, a negatively charged polysaccharide (Section 11.3) found in mast cells near the walls of blood vessels and on the surfaces of endothelial cells (Figure 10.33). Heparin acts as an anticoagulant by increasing the rate of formation of irreversible complexes between antithrombin III and the serine protease clotting factors.

The importance of the ratio of thrombin to antithrombin is illustrated in the case of a 14-year-old boy who died of a bleeding disorder because of a mutation in his α₁-antitrypsin, which normally inhibits elastase. Methionine 358 in α₁-antitrypsin’s binding pocket for elastase was replaced by arginine, resulting in a change in specificity from an elastase inhibitor to a thrombin inhibitor. Activity of α₁-anti-trypsin normally increases markedly after injury to counteract excess elastase arising from stimulated neutrophils. The mutant α₁-antitrypsin caused the patient’s thrombin activity to drop to such a low level that hemorrhage ensued. We see here a striking example of how a change of a single residue in a protein can dramatically alter specificity and an example of the critical importance of having the right amount of a protease inhibitor.

Antithrombin limits the extent of clot formation, but what happens to the clots themselves? Clots are not permanent structures but are designed to dissolve when the structural integrity of damaged areas is restored. Fibrin is degraded by plasmin, a serine protease that hydrolyzes peptide bonds in the coiled-coil regions. Plasmin molecules can diffuse through aqueous channels in the porous fibrin clot to cut the accessible connector rods. Plasmin is formed by the proteolytic activation of plasminogen, an inactive precursor that has a high affinity for the fibrin clots. This conversion is carried out by tissue-type plasminogen activator (TPA), a 72-kDa protein that has a domain structure closely related to that of prothrombin (Figure 10.34). However, a domain that targets TPA to fibrin clots replaces the membrane-targeting gla domain of prothrombin. The TPA bound to fibrin clots swiftly activates adhering plasminogen. In contrast, TPA activates free plasminogen very slowly. The gene for TPA has been cloned and expressed in cultured mammalian cells. TPA administered at the onset of a heart attack or a stroke caused by a blood clot increases the likelihood of survival without physical or cognitive disabilities (Figure 10.35).

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Some important breakthroughs in the elucidation of clotting pathways have come from studies of patients with bleeding disorders. Classic hemophilia, or hemophilia A, is the best-known clotting defect. This disorder is genetically transmitted as a sex-linked recessive characteristic. In classic hemophilia, factor VIII (antihemophilic factor) of the intrinsic pathway is missing or has markedly reduced activity. Although factor VIII is not itself a protease, it markedly stimulates the activation of factor X, the final protease of the intrinsic pathway, by factor IX_a, a serine protease (Figure 10.36). Thus, activation of the intrinsic pathway is severely impaired in hemophilia.

In the past, hemophiliacs were treated with transfusions of a concentrated plasma fraction containing factor VIII. This therapy carried the risk of infection. Indeed, many hemophiliacs contracted hepatitis and, more recently, AIDS. A safer source of factor VIII was urgently needed. With the use of biochemical purification and recombinant DNA techniques, the gene for factor VIII was isolated and expressed in cells grown in culture. Recombinant factor VIII purified from these cells has largely replaced plasma concentrates in treating hemophilia.