Serine proteinase, activity

As with all complex biological systems we should not forget the close interplay between oxidative and proteolytic systems (see Fig. 2). For example, it has been shown that at a localised inflammatory site, oxidative inactivation of protease inhibitors may lead to a proteolytic cascade resulting in down-stream MMP activation through the localised action of serine proteinases activating previously latent MMPs (see Fig. 2). Equally, the generation of active MMPs (post-oxidant exposure) may be involved in the site-specific catalytic inactivation of serine-protease inhibitors [59] at an inflammatory site with the consequent generation of an elevated serine protease load and connective tissue proteolysis (see Fig. 2). 

Klingel, S Rothe, G., Kellermann, W., and Valet, G. (1994) Flow cytometric determination of cysteine and serine proteinase activities in living cells with rhodamine 110 substrates. Methods Cell Biol. 41,449 459. 

Mechanisms of Serine Proteinase Activation Insights for the Development of Biopharmaceuticals for Coagulation and Fibrinolysis... 

Figure 6.23 Schematic diagram illustrating the active site loop regions (red) in three forms of the serpins. (a) In the active form the loop protrudes from the main part of the molecuie poised to interact with the active site of a serine proteinase. The first few residues of the ioop form a short p strand inserted between ps and pis of sheet A. (h) As a result of inhibiting proteases, the serpin molecules are cleaved at the tip of the active site ioop region, in the cleaved form the N-terminal part of the loop inserts itself between p strands 5 and 15 and forms a long p strand (red) in the middie of the p sheet, (c) In the most stable form, the latent form, which is inactive, the N-terminai part of the ioop forms an inserted p strand as in the cleaved form and the remaining residues form a ioop at the other end of the p sheet. (Adapted from R.W. Carreii et ai., Structure 2 257-270, 1994.)...

A closer examination of these essential residues, including the catalytic triad, reveals that they are all part of the same two loop regions in the two domains (Figure 11.10). The domains are oriented so that the ends of the two barrels that contain the Greek key crossover connection (described in Chapter 5) between p strands 3 and 4 face each other along the active site. The essential residues in the active site are in these two crossover connections and in the adjacent hairpin loops between p strands 5 and 6. Most of these essential residues are conserved between different members of the chymotrypsin superfamily. They are, of course, surrounded by other parts of the polypeptide chains, which provide minor modifications of the active site, specific for each particular serine proteinase. 

Serine proteinases such as chymotrypsin and subtilisin catalyze the cleavage of peptide bonds. Four features essential for catalysis are present in the three-dimensional structures of all serine proteinases a catalytic triad, an oxyanion binding site, a substrate specificity pocket, and a nonspecific binding site for polypeptide substrates. These four features, in a very similar arrangement, are present in both chymotrypsin and subtilisin even though they are achieved in the two enzymes in completely different ways by quite different three-dimensional structures. Chymotrypsin is built up from two p-barrel domains, whereas the subtilisin structure is of the a/p type. These two enzymes provide an example of convergent evolution where completely different loop regions, attached to different framework structures, form similar active sites. 

Proteinase-activated recqrtors (PARs) are a unique family of G-protein-coupled receptors (GPCRs) that are activated in response to serine proteinases. There are four PAR family members PAR-1 through to PAR-4. PAR-1 and PAR-3 respond to thrombin, PAR-2 responds to trypsin, whilst PAR-4 is sensitive to both thrombin- and trypsin-related proteinases. 

Proteinase-activated Receptors. Figure 1 Activation of proteinase-activated receptors (PARs) through proteolytic cleavage with serine proteinases (1) and independent of cleavage though PAR-specific activating peptides (2). 

A tethered ligand is the new N-terminal formed following serine proteinase-mediated cleavage of the original N-terminal of a PAR family member, which is responsible for activation of the receptor. 

Heparin cofactor II, when activated by binding to glycosaminoglycans (dermatan sulfate, heparins, and heparin), inhibits thrombin (24). The 43-kDa serpin, proteinase nexin 1, possesses 30% sequence homology with ATIII and can be activated by binding to heparin to inhibit several serine proteinases including thrombin (25). Proteinase nexin 2 is found within the platelet a-granule and is released when platelets are activated (26). It is able to inhibit factor XIa. 

A compound that reduces the activity of an enzyme is known as an inhibitor. Inhibitors are usually small molecules but some are peptides or proteins. For example, there are a number of proteolytic enzymes in the blood that have serine in their active site. If the activities of these enzymes are too high, they can cause problems. Consequently, inhibitor proteins, known as serine proteinase inhibitors (serpins), are present in blood indeed, about 10% of all the plasma proteins are serpins (Box 3.4). 

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). 

The catalytic mechanism in this class is based upon similar chemical principles as the mechanism of the serine proteinases. A cysteine residue in the active site is activated by a histidine imidazolium side chain and carries out a nucleophilic attack on the carbonyl carbon of the scissile peptide bond with the complex going through an acyl intermediate transition state (28,29). Certain members of this class of enzymes have pH optima in the acidic range and... 

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