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Protease inhibitors

Submitted to: Dr. Sashi Madaan Dapartment of Biochemistry

Submitted by: Punesh Ph.D. Biochemistry

INTRODUCTION Protein turnover is an important process in living systems. Proteins that have served their purpose must be degraded so that their constituent amino acids can be recycled for the synthesis of new proteins. Proteins ingested in the diet must be broken down into small peptides and amino acids for absorption in the gut. Furthermore, proteolytic reactions are important in regulating the activity of certain enzymes and other proteins. Proteases cleave proteins by a hydrolysis reaction the addition of a molecule of water to a peptide bond:

Although the hydrolysis of peptide bonds is thermodynamically favored, such hydrolysis reactions are extremely slow. In the absence of a catalyst, the half-life for the hydrolysis of a typical peptide at neutral pH is estimated to be between 10 and 1000 years. Yet, peptide bonds must be hydrolyzed within milliseconds in some biochemical processes. The inhibitors are proteins that interact with proteases and suppress their proteolytic activity. Such inhibitors are widespread in plant tissues and specific for one of the four types of proteases (serine, cysteine, and aspartyl proteases and metalloproteases). We focus on inhibitors of serine and cysteine proteases, because these inhibitors prevail in plants and, on the other hand, their target enzymes are most common in insects. Protein inhibitors of proteinases are widely distributed in higher plants. These enzymes were mostly isolated from leguminous seeds. Soybean inhibitors were the most thoroughly examined with respect to their structures and mechanisms of interaction with proteases. Various inhibitors were isolated from cereal seeds (wheat, rice, and maize). The great interest in studies of proteinase inhibitors from seeds is primarily related to their possible involvement in the regulation of protein synthesis and degradation, as well as in plant defenses against insects and microorganisms. In addition, proteinase inhibitors attract the attention of researchers as substances capable of influencing the nutritional value of proteins for humans and animals. Serine and cysteine protease inhibitors are mostly small proteins, 6–25 kDa in molecular weight. Their content in seeds reaches 4–10% of the total water-soluble protein. The protective role of such inhibitors is supported by their preferential association with seeds, which are most attractive to various pests and most vulnerable because of their metabolic inactivity and the consequent lack of an active defense system. Moreover, inhibitors are highly stable under unfavorable conditions (extreme pH, higher temperatures, etc.). Another argument in favor of the protective role of protease inhibitors is that inhibitors originating from the same source greatly vary in specificity. It is noteworthy that most inhibitors exert no effect on cognate seed proteases and highly efficiently inhibit animal and bacterial proteolytic enzymes as well as proteases of the digestive tract of insect pests. An important field of research is seeking for new inhibitors specific for major digestive proteases of insects. Studies of the Colorado beetle digestive complex showed that aspartyl protease starts hydrolysis of the protein component of the food. Hence, expression of a gene for its inhibitor in potato is potentially more effective

than expression of the oryzacystatin gene and may play a crucial role in suppressing insect digestion Interactions of proteases with inhibitors: Proteolytic activation of zymogens is an irreversible process. This creates a need for additional mechanisms regulating post-translationally activated proteases. Modulation by specific inhibitors is the most common of these. However, in bacteria, endogenous proteins inhibiting the proteases are relatively rare. Higher organisms have developed various inhibitors of proteolytic enzymes, not only to control self-produced proteases. Protease inhibitors are also an important element of the host anti-pathogen defence system. Mammal defence protease inhibitors belong to two classes: the active-site inhibitors, represented by a superfamilies of serpins and cystatins; and the α2macroglobulins. The members of the former group inactivate enzymes by binding to the active site, the latter act as molecular traps for the proteases. However, some bacteria have learned to utilize the inhibitors to regulate the activity of their extracellular proteases for their own purposes. Protease Inhibitors Are Important Drugs: Compounds that block or modulate the activities of proteases can have dramatic biological effects. Most natural protease inhibitors are similar in structure to the peptide substrates of the enzyme that each inhibits. Several important drugs are protease inhibitors. For example, captopril, an inhibitor of the metalloprotease angiotensinconverting enzyme (ACE), has been used to regulate blood pressure. Crixivan, an inhibitor of the HIV protease, is used in the treatment of AIDS. This protease cleaves multidomain viral proteins into their active forms; blocking this process completely prevents the virus from being infectious. To prevent unwanted side effects, protease inhibitors used as drugs must be specific for one enzyme without inhibiting other proteins within the body. Crixivan is constructed around an alcohol that mimics the tetrahedral intermediate; other groups are present to bind into the S2, S1, S1 , and S2 recognition sites on the enzyme. The results of x-ray crystallographic studies revealed the structure of the enzyme-Crixivan complex, showing that Crixivan adopts a conformation that approximates the twofold symmetry of the enzyme. The active site of HIV protease is covered by two apparently flexible flaps that fold down on top of the bound inhibitor. The hydroxyl group of the central alcohol interacts with two aspartate residues of the active site, one in each subunit. In addition, two carbonyl groups of the inhibitor are hydrogen bonded to a water molecule (not shown), which, in turn, is hydrogen bonded to a peptide NH group in each of the flaps. This interaction of the inhibitor with water and the enzyme is not possible with cellular aspartyl proteases such as renin and thus may contribute to the specificity of Crixivan and other inhibitors for HIV protease. Specific Inhibitors of Some Proteolytic Enzymes: 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 stop proteolysis. Specific protease inhibitors accomplish this task. For example, pancreatic trypsin inhibitor, a 6-kd

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 -18 kcal mol-1 (-75 kJ 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 showed 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. 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 and is turned over slowly. Why does trypsin inhibitor exist? Indeed, the amount of trypsin is much greater that that of the inhibitor. Under what circumstances is it beneficial to inhibit trypsin? Consequently, it is vital that even small amounts of trypsin be prevented from initiating the cascade prematurely. Trypsin molecules activated in the pancreas or pancreatic ducts could severely damage those tissues, leading to acute pancreatitis. Tissue necrosis may result from the activation of proteolytic enzymes (as well as prolipases) by trypsin, and hemorrhaging may result from its activation of elastase. We see here the physiological need for the tight binding of the inhibitor to trypsin. Pancreatic trypsin inhibitor is not the only important protease inhibitor. a 1 -Antitrypsin (also called a 1 -antiproteinase), a 53-kd plasma protein, 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, a 1-antitrypsin blocks the action of target enzymes by binding nearly irreversibly to their active sites. Genetic disorders leading to a deficiency of a 1antitrypsin show that this inhibitor is physiologically important. 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 destructive lung disease). 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, 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. There is another protease inhibitor, antithrombin III, which is involved in the control of blood clotting. Plant Protease inhibitors: Many authors pointed out that protein inhibitors of proteolytic enzymes are important factors of plant defense, primarily against insects and phytopathogenic microorganisms. Despite certain common features, responses of plants to injury inflicted by insects and pathogenic microorganisms are different. These differences are found in the mechanisms of signal transduction and certain features of responses. Thus, it is expedient to consider separately the involvement of inhibitors of proteolytic enzymes in plant defenses against insects and pathogenic microorganisms. The first data suggesting that inhibitors of proteolytic enzymes are involved in plant defenses against insects were obtained in the mid-1960s: proteins acting as specific inhibitors of intestinal proteolytic enzymes of insects of the genus Tribolium, which cause severe losses of grain and grain processing products, were isolated from soy and wheat seeds ( Triticum aestivum L.). Another important line of evidence comes from experiments with tomato and potato leaves; their mechanical injury or damage done by Colorado potato beetle (Leptinotarsa decemlineata Say.) caused systemic induction of serine protease inhibitors. Systemic induction of proteinase inhibitors by plant tissue injury was most extensively studied in experiments with representatives of the family Solanacea, tomatoes, potato (Solanum tuberosum L.), and tobacco. Plant protease inhibitors family: 1. Family of the Baumann–Birk inhibitor: A soybean protein distinct from SKTI was first described by Baumann in 1946 and characterized in detail by Birk et al.. A molecule of this protein, comprising 71 amino acid residues, contains seven disulfide bonds and has a molecular weight of 8 kDa. The ability to bind trypsin and chymotrypsin at the same time, with the formation of ternary complexes, is a characteristic feature of this inhibitor. The molecule of the inhibitorn is composed of two homologous domains. 2. Serpins: Serpins constitute a unique family of serine proteinase inhibitors, the members of which are found in plants, animals, and viruses. The mechanism whereby serpins interact with the enzymes differs from the classic interaction characteristic of all other serine proteinase inhibitors of plant origin. Specifically, it involves cleavage of the peptide bond P1–P1' in the molecule of the inhibitor and the formation of a covalent bond with the Ser residue in the active site of the proteinase. The process is associated with considerable conformational changes of the reacting molecules, which slows down the rate of deacylation and results in the formation of stable inhibitor–enzyme complexes 3. Phytocystatins: Inhibitors of cysteine proteinases, acting on enzymes of the papain family, are the best studied among plant proteinase inhibitors of other families. The majority of proteins of this family are represented by members of the cystatin superfamily (within which they constitute a specific family of phytocystatins). The molecule of each phytocystatin contains all three structural elements involved in the formation of complexes with enzymes in animal cystatins. These elements include the

residue Gly, which is located in the N-terminal portion of the molecule, the conserved amino acid sequence Gln–X–Val–X–Gly, and the residue Trp in the C-terminal portion of the molecule. The conserved amino acid sequence Gln–X–Val–X–Gly seems to make the major contribution to the formation of the complex with a susceptible proteinase. This conclusion is supported by the observation that phytocystatins devoid of Gly and Trp residues in appropriate positions may retain relatively high inhibitory activities. NMR studies of the spatial structure of rice cystatin (oryzacystatin I) demonstrated that all three elements involved in the formation of the complex with the enzyme take the same positions as in egg white cystatin. In addition, phytocystatins contain another conserved sequence, Leu–Ala–Arg–Phe–Ala–Val–X–Glu–His–Asn, in the vicinity of the Nterminal α-helix, which is absent from cystatins of animal origin. The role of this sequence has not been clarified thus far. Its presence may reflect ancillary functions played by cystatins in plants, which may have no immediate relation to effects on proteinases. 4. Mustard trypsin inhibitor family: The seeds of cruciferous plants (Cruciferae) contain thermostable low molecular weight trypsin inhibitors. In the seed of rape (Brassica napus L.), these inhibitors account for 60–85% of total trypsin-inhibiting activity. The first inhibitor to be obtained with a high degree of purity (MTI-2) was isolated from the seed of white mustard (Sinapis alba L.). MTI-2 has a molecular weight of 7 kDa; its molecule contains 63 amino acids, including eight Cys residues forming four disulfide bonds. An inhibitor with closely similar properties (RTI) was isolated from the seed of oil rape (Brassica napus (L.) var. oleifera). MTI-2 and RTI were shown to have homologous primary structures (70% amino acid residues are shared), which differed from any other structures of plant inhibitors of serine proteinases known at that time. Subsequent studies demonstrated the presence of several forms of the inhibitors in rape and mustard seeds 5. Family of trypsin and α-amylase inhibitors from cereal grains: Trypsin inhibitors, which account for 5−10% of total water-soluble protein in barley, wheat, and rye seeds, are classified with two different families. The inhibitors of the germ portion of caryopsis belong to the Baumann–Birk family (described above), whereas the inhibitors contained in the endosperm constitute a separate family, which embraces the inhibitors of trypsin and heterologous α-amylases. Trypsin inhibitors of these two types differ in both the location within the caryopsis and the behavior throughout seed development and germination 6. Potato inhibitor I family: Chymotrypsin inhibitor I was isolated from potato tubers and crystallized in 1963. In addition to chymotrypsin, this protein acts on microbial serine proteinases and inhibits trypsin (even though weakly). The inhibitor has a molecular weight of 41 kDa; it is an oligomer composed of five structurally similar protomers. 7. Potato inhibitor II family: The first representatives of this family were isolated from potato tubers and tomato leaves. These proteins act as potent inhibitors of chymotrypsin and subtilisin, their effects on trypsin being less pronounced. The molecular weight of each inhibitor approximates 21 kDa; the molecules are dimers of similar but not completely identical subunits.

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