Proteinase

There is abundant evidence that a diverse array of pro-teinases, particularly those of macrophage or leucocyte origin, have a fundamental role in the injury processes associated with diseases such as ai-antitrypsin deficiency, smoking-related emphysema, cystic fibrosis, bronchiectasis, bronchitis and other respiratory syndromes (Karlinsky and Snider, 1978 Stockley, 1983 Senior and Campbell, 1983 Surer etal., 1984 Janoff, 1985 Snider etal., 1991). However, there has been little exploration of their potential involvement in other airway disorders that have an inflammatory cell aetiology, such as certain types of specialized fibrotic disorders including chronic severe asthma. This is particularly true for the matrix metalloproteinases (MMPs), despite the expectation from their individual substrate specificities (Emonard and 

Grimaud, 1990) that they should be of fundamental importance in diseases associated with remodelling and fibrosis of the basement membrane, epithelial injury and fibrosis. 

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Hydrolases. Enzymes catalysing the hydrolytic cleavage ofC —O, C —N and C —C bonds. The systematic name always includes hydrolase but the recommended name is often formed by the addition of ase to the substrate. Examples are esterases, glucosidases, peptidases, proteinases, phospholipases. Other bonds may be cleaved besides those cited, e.g. during the action of sulphatases and phosphatases. 

McPhalen, C. A., James, M. N. G. Structural comparison of two serine proteinase-protein inhibitor complexes Eglin-C-Subtilisin Carlsberg and CI-2-subtilisin novo. Biochemistry 27 (1988) 6582-6598... 

Hansson, T., Aqvist, J. Estimation of binding free energies for HIV proteinase inhibitors by molecular dynamics simulations. Prot. Eng. 8 (1995) 1137-1144... 

Hansson T and J Aqvist 1995. Estimation of Binding Free Energies for HIV Proteinase Inhibitors b Molecular Dynamics Simulations. Protein Engineering 8 1137-1144. 

Protein acidulant Protein additives Protein ammo acids a-l-Proteinase inhibitor Protein-based mimetics Protein Ca [42617-41-4] Protein channels Protein chromatography Protein crystal growth... 

Factor VIII, immunoglobulin, and albumin are all held as protein precipitates, the first as cryoprecipitate and the others as the Cohn fractions FI + II + III (or FII + III) and FIV + V (or FV), respectively (Table 7, Fig. 2). Similarly, Fractions FIVj + FIV can provide an intermediate product for the preparation of antithrombin III and a-1-proteinase inhibitor. This abiUty to reduce plasma to a number of compact, stable, intermediate products, together with the bacteriacidal properties of cold-ethanol, are the principal reasons these methods are stiU used industrially. 

IV, a-l-proteinase inhibitor antithrombin III IgM ceruloplasmin a- and P-globutins a-tipoprotein albumin 5-10... 

Alpha-1-proteinase inhibitor and antithrombin III are used to treat people with hereditary deficiencies of these proteins. Both can be recovered from Cohn Fraction IV (Table 7) using ion-exchange chromatography (52) and affinity chromatography (197), respectively. Some manufacturers recover antithrombin III directiy from the plasma stream by affinity adsorption (56,198,199). 

Figure 2.19 Organization of polypeptide chains into domains. Small protein molecules like the epidermal growth factor, EGF, comprise only one domain. Others, like the serine proteinase chymotrypsin, are arranged in two domains that are required to form a functional unit (see Chapter 11). Many of the proteins that are involved in blood coagulation and fibrinolysis, such as urokinase, factor IX, and plasminogen, have long polypeptide chains that comprise different combinations of domains homologous to EGF and serine proteinases and, in addition, calcium-binding domains and Kringle domains.

Serine proteinase domains that are homologous to chymotrypsin, which has about 245 amino acids arranged in two domains. 

In the first edition of this book this chapter was entitled "Antiparallel Beta Structures" but we have had to change this because an entirely unexpected structure, the p helix, was discovered in 1993. The p helix, which is not related to the numerous antiparallel p structures discussed so far, was first seen in the bacterial enzyme pectate lyase, the stmcture of which was determined by the group of Frances Jurnak at the University of California, Riverside. Subsequently several other protein structures have been found to contain p helices, including extracellular bacterial proteinases and the bacteriophage P22 tailspike protein. 

In these p-helix structures the polypeptide chain is coiled into a wide helix, formed by p strands separated by loop regions. In the simplest form, the two-sheet p helix, each turn of the helix comprises two p strands and two loop regions (Figure 5.28). This structural unit is repeated three times in extracellular bacterial proteinases to form a right-handed coiled structure which comprises two adjacent three-stranded parallel p sheets with a hydrophobic core in between. 

Serpins inhibit serine proteinases with a spring-loaded safety catch mechanism... 

Serpins form very tight complexes with their corresponding serine pro-teinases, thereby inhibiting the latter. A flexible loop region of the serpin binds to the active site of the proteinases. Upon release of the serpin from the complex its polypeptide chain is cleaved by the proteinase in the middle of this loop region and the molecule is subsequently degraded. In addition to the active and cleaved states of the serpins there is also a latent state with an intact polypeptide chain that is functionally inactive and does not bind to the proteinase. 

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.)...

Schreuder, H.A., et al. The intact and cleaved human antithrombin III complex as a model for serpin-proteinase interactions. Nature (Struct. Biol.)... 

In this chapter we shall illustrate some fundamental aspects of enzyme catalysis using as an example the serine proteinases, a group of enzymes that hydrolyze peptide bonds in proteins. We also examine how the transition state is stabilized in this particular case. 

All the well-characterized proteinases belong to one or other of four families serine, cysteine, aspartic, or metallo proteinases. This classification is based on a functional criterion, namely, the nature of the most prominent functional group in the active site. Members of the same functional family are usually evolutionarily related, but there are exceptions to this rule. We... 

Serine proteinases cleave peptide bonds by forming tetrahedral transition states... 

The serine proteinases have been very extensively studied, both by kinetic methods in solution and by x-ray structural studies to high resolution. From all these studies the following reaction mechanism has emerged. 

Figure 11.4 Serine proteinases catalyze the hydrolysis of peptide bonds within a polypeptide chain. The bond that is cleaved is called the scissile bond. (Ra) and (Rb)j/ represent polypeptide chains of varying lengths.

Figure 11.6 A schematic view of the presumed binding mode of the tetrahedral transition state intermediate for the deacylation step. The four essential features of the serine proteinases are highlighted in yellow the catalytic triad, the oxyanion hole, the specificity pocket, and the unspecific main-chain substrate binding.

Four important structural features are required for the catalytic action of serine proteinases... 

The serine proteinases have four important structural features that facilitate this mechanism of catalysis (Figure 11.6). 

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