Proteinase inactivation

Proteinase Inactivation Antithrombin Deficiency-homozygous-only with 1 per 5,000 to 1 125 2.3 Liver 61-92 lANT, lATH, ANT3 HUMAN... 

Proteinase inactivation occurs by a stoichiometric reaction between proteinase and inhibitor that results in the formation of a covalent ester bond between the reactive site residue of the inhibitor (Arg in antithrombin) and the active site residue (Ser in the proteinase). The proteinases thrombin, factor Xa, factor IXa, and, less effectively, factor Vila and factor XIa are all inactivated by antithrombin (Figure 36-16). Other SERPINS can inactivate procoagulant proteinases, heparin cofactor II can inactivate thrombin, and O I-proteinase inhibitor can inactivate factor Xa. An altered a i -proteinase inhibitor (a i -proteinase inhibitor... 

The fibrinolytic system removes the fibrin of the hemostatic plug and thus is responsible for the temporary existence of the fibrin clot. The proteolytic action of plasmin on fibrin and fibrinogen is extensive and more like the digestive proteolysis catalyzed by trypsin and chy-motrypsin than the proteolysis involved in proteinase precursor activation. The fibrinolytic subsystem includes the reactions of plasminogen activation, plasmin inactivation, and fibrin digestion. As is common throughout the hemostatic system, irreversible activation reactions of the fibrinolytic system are opposed by irreversible proteinase inactivation. 

The role of therapeutic inhibitors of elastase is to prevent the degradation of extracellular matrix proteins following the release of elastase from activated neutrophils. To determine which inhibitors of elastase may be of therapeutic value, there are several properties that must be considered. These include rate of proteinase inactivation, enzyme selectivity, size, susceptibility to inactivation, and their potential for antigenicity. Some of these properties are reviewed in the following section. 

Proteases (proteinases, peptidases, or proteolytic enzymes) are enzymes that break peptide bonds between amino acids of proteins. The process is called peptide cleavage, a common mechanism of activation or inactivation of enzymes. They use a molecule of water for this, and are thus classified as hydrolases. 

In some cases, receptor inactivation, e.g., of the V2 vasopressin receptor, is mediated by agonist-induced enzymatic cleavage of the GPCR. This nonendocytic proteolysis is promoted by a plasma membrane-associated metalloprotease. Proteinase-activated receptors (PARs) such as the thrombin receptor also follow a distinctly different pathway. PARs require the enzymatic cleavage of their N terminus, and the newly generated N terminus activates the receptor. Once... 

Allaire M, Chernaia MM, Malcolm BA, James MN (1994) Picomaviral 3C cysteine proteinases have a fold similar to chymotrypsin-Kke serine proteinases. Nature 369 72-76 Altman MD, Nalivaika EA, Prabu-Jeyabalan M, Schiffer CA, Tidor B (2008) Computational design and experimental study of tighter binding peptides to an inactivated mutant of HIV-1 protease. Proteins 70 678-694... 

KROGDAHL A, HOLM H (1981) Soybean proteinase inhibitors and human proteolytic enzymes selective inactivation of inhibitors by treatment with human gastric juice. /M/fr. Ill 2045-51. 

Desrochers, P.E. and Weiss, S.J. (1988). Proteolytic inactivation of alpha-l-proteinase inhibitor by a neutrophil metalloproteinase. J. Clin. Invest. 81, 1646-1650. 

Wakselman, M. Xie, J. Mazaleyrat, J.-P. Boggetto, N. Vilain, A. C. Montagne, J.-J. Reboud-Ravaux, M. New mechanism-based inactivators of trypsin-like proteinases. Selective inactivation of urokinase by functionalized cyclopeptides incorporating a sulfoniomethyl-substituted mera-aminobenzoic acid residue. J. Med. Chem. 1993, 36, 1539-1547. 

Proteinases and antiproteinases are part of the normal protective and repair mechanisms in the lungs. The imbalance of proteinase-antiproteinase activity in COPD is a result of either increased production or activity of destructive proteinases or inactivation or reduced production of protective antiproteinases. AAT (an antiproteinase) inhibits trypsin, elastase, and several other proteolytic enzymes. Deficiency of AAT results in unopposed proteinase activity, which promotes destruction of alveolar walls and lung parenchyma, leading to emphysema. 

Some legumes, including raw soy or peanut flour are known to contain certain antinutritional factors such as proteinase inhibitors and hemagglutinins or lectins (21,22). These factors can be inactivated, for the most part, by moist heat, during processing. Interestingly, peanut flour contained more trypsin inhibitor and lectin than did soy flour (22). 

C5a is inactivated by the myeloperoxidase-H202 system, which oxidises a methionine residue (Met 70) on the molecule group A streptococcal endo-proteinases also abolish chemotactic activity of C5a and related compounds. Neutrophil lysosomal enzymes (e.g. elastase and cathepsin G) also destroy C5a chemotactic activity, but as these proteases are inhibited by the serum antiproteinases, a -antiproteinase and a2-macroglobulin, the physiological role of neutrophilic proteases in the inactivation of C5a is questionable. Two chemotactic factor inactivators have been found in human serum an a-globulin that specifically and irreversibly inactivates C5-derived chemotactic factors, and a / -globulin that inactivates bacterial chemotactic factors. These activities are heat labile (destroyed by treatment at 56 °C for 30 min) and are distinct from those attributable to anaphylatoxin inactivator. An apparently specific inhibitor of C5-derived chemotactic activity has also been described in human synovial fluid and peritoneal fluid. This factor (molecular mass of 40 kDa) is heat stable and acts directly on C5a. 

The role of the fibrinolytic system is to dissolve any clots that are formed within the intact vascular system and so restrict clot formation to the site of injury. The digestion of the fibrin and hence its lysis is catalysed by the proteolytic enzyme, plasmin, another serine proteinase. Plasmin is formed from the inactive precursor, plasminogen, by the activity of yet other proteolytic enzymes, urokinase, streptokinase and tissue plasminogen activator (tPA) which are also serine proteinases. These enzymes only hydrolyse plasminogen that is bound to the fibrin. Any plasmin that escapes into the general circulation is inactivated by binding to a serpin (Box 17.2). 

To prevent self-digestion, the pancreas releases most proteolytic enzymes into the duodenum in an inactive form as proenzymes (zymogens). Additional protection from the effects of premature activation of pancreatic proteinases is provided by proteinase inhibitors in the pancreatic tissue, which inactivate active enzymes by complex formation (right). 

The fibrin thrombus resulting from blood clotting (see p. 290) is dissolved again by plasmin, a serine proteinase found in the blood plasma. For this purpose, the precursor plasminogen first has to be proteolyti-cally activated by enzymes from various tissues. This group includes the plasminogen activator from the kidney (urokinase) and tissue plasminogen activator (t-PA) from vascular endothelia. By contrast, the plasma protein a2-antiplasmin, which binds to active plasmin and thereby inactivates it, inhibits fibrinolysis. 

A potent irreversible inhibitor (abbreviated DFP) of many serine proteinases and serine esterases (especially acetylcholinesterase). This substance is EXTREMELY POISONOUS, but the vapor state can be minimized by using dry, water-miscible solvents such as 2-propanoL. Aqueous solutions become inactivated by hydrolysis, but solutions made with dry 2-propanol are stable at -20°C for many months. 

Cathepsin D (EC3.4.23.5). It has been known for more than 20 years that milk also contains an acid proteinase, (optimum pH ss 4.0) which is now known to be cathepsin D, a lysozomal enzyme. It is relatively heat labile (inactivated by 70°C x 10 min). Its activity in milk has not been studied extensively and its significance is unknown. At least some of the indigenous acid proteinase is incorporated into cheese curd its specificity on asl- and / -caseins is quite similar to that of chymosin but it has very poor milk-clotting activity (McSweeney, Fox and Olson, 1995). It may contribute to proteolysis in cheese but its activity is probably normally overshadowed by chymosin, which is present at a much higher level. 

Figure 9.12 Thermal inactivation of Ps. fluorescens AFT 36 proteinase on heating for 1 min in 0.1 M phosphate buffer, pH 6.6 (O) or in a synthetic milk salts buffer, pH 6 ( ) (from Stepaniak, Fox and Daly, 1982).

Driessen, F.M. (1989) Inactivation of lipases and proteinases (indigenous and bacterial), in Heat-induced Changes in Milk (ed. P.F. Fox), Bulletin 238, International Dairy Federation, Brussels, pp. 71-93. 

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