Proteinase active center

Metalloproteinases are a subgroup of proteinases. They are responsible for the cleavage of peptide bonds within a protein (proteolysis). Metalloproteinases contain a metal ion in the active center and are divided into four subclasses dependent on their mechanism of catalysis. 

A large group of proteinases contain serine in their active center. The serine proteases include, for example, the digestive enzymes trypsin, chymotrypsin, and elastase (see pp. 94 and 268), many coagulation factors (see p. 290), and the fibrinolytic enzyme plos-min and its activators (see p. 292). 

Cysteine proteinase contain a catalytic ally active cysteine snUhy ljtQup (Cys-25) and a histidine group (His-i59) within the active center of the sizyme [51]. Alkylation of the active-center sulfhydryl group renders the product inactive. The minimal reaction mechanism is represented in Figure 4 [52]. 

El is well known that the cysteine proteinase of the papaya latex differ in their ability to hydrolyze proteins and synthetic peptides. Schechter and Beiger [67] first demonstrated that the landing regions in the active center of proteinases can be divided into different subtitles, of whidi die Sz subsite seems to be the most important in papain. 

Many excellent reviews have already been written on the subject of the catalytic centers of serine and thiol proteinases (e.g.. Kraut, 1977 Baker and Drenth, 1987 Warshel etai, 1989). In this paper the focus is specifically on the structure of the catalytic triad in lipases, with emphasis on the differences from and similarities to the catalytic centers of proteinases. The atomic coordinates for the G. candidum lipase were not available when this review was written, and the analysis of the stereochemistry of the active centers is therefore restricted to lipases from R. miehei and the human pancreas. 

In the past, considerable significance was attached to the syn-carboxylate interaction. Candour (1981) suggested that the syn electron pair may be more basic than the anti pair by a factor 10 -10. This seemed to explain the enhanced basicity of imidazole. However, studies of databases (Allen and Kirby, 1991) and model compounds (Zimmerman et ai, 1991) indicate that the difference in the relative basicities between syn- and anti-carboxylates is marginal (0.4—0.6 units). The hPL triad and the Asp-His couple in the active center of phospholipase A2 appear to support this view. Thus, the preference toward syn-type bonds observed in most serine proteinases may be due to packing effects in the active centers rather than to stereoelectronic effects. 

A reduction of the proteinase inhibitory activity is due to oxidation of the reactive methionine residue of the active center by the cigarette smoke as well as by oxygen radicals produced by leukocytes and macrophages. Emphysema is a common disease, and its most common cause is cigarette smoking. Only 1-2% of cases are due to genetic deficiency of apAT. 

Specificity is often described in terms of the subsite model of Schechter and Berger [67,68] (Fig. 6). In terms of this model, the substrate may be represented as P4-P3-P2-Pi-T-P i-P,2, where P represents an amino acid residue that binds to a particular region in the active center (subsite Si.S i, etc.) and the arrow indicates the scissible bond, of which Pi provides the carbonyl moiety and P i the amino moiety. The reactivity and inhibition studies of Berger and Schechter [68] showed that papain has a strong affinity for Phe, Tyr, Val, or Leu at P2 of the substrate. A similar preference is shown by all papaya proteinases, although significant differences exist. 

It is well understood that caspases are critical for the process of apoptosis. Caspases are cysteinyl-containing active center proteases with specificity for protein cleavage after aspartyl residues. Thus the term caspase is derived from (ysteinyl-containing aij artate-specific proteinase. The caspases are responsible for many of the hallmarks of apoptosis defined in Table 18.1, mediated through their cleavage of specific polypeptide substrates. The caspases (e.g., caspase-2, -3, -6, -7, -8, -9, -10, and -12 in the mouse) are not the only proteases involved in PCD, as calpains have also been shown to play... 

A second elastolytic enzyme (Af, 21,900) has been isolated from porcine pancreas. It shows higher activity than chymotrypsin in the hydrolysis of acetyltyrosine ester, which is used routinely to assay chymotrypsin. Another E,-like enzyme, a-lytic proteinase (Af, 19,900, 198 amino acids) has been isolated from the soil bacterium Myxobacter 495. This enzyme is remarkably similar to pancreatic E. both in structure (41 % homology, sequence in the active center Gly-Asp-Ser-Gly, 3 homologous disulfide bridges) and substrate specificity. Another E. (Af, 22,300) has been isolated from Pseudomonas aeruginosa. 

Subtilisin (EC 3.4.21.4) an extracellular, single chain, alkaline serine protease from Bacillus subtilis and related species. S. are known from four different species of Bacillus S. Carlsberg (274 amino acid residues, M, 27,277), S. BPN (275 amino acid residues, M, 27,537), S. Novo (identical with S.BPN ) and S. amylosacchariticus (275 amino acid residues, M, 27671). The observed sequence differences between different S. represent conservative substitutions and are limited to the surface amino acids. Like the pancreatic proteinases, S. has catalytic Ser22i, His64 and Asnjj residues, but it is structurally very different from the other serine proteases, e. g. the active center of S. is -Thr-Ser-Met-, whereas that of the pancreatic enzymes is -Asp-Ser-Gly- pancreatic enzymes contain 4- disulfide bridges, whereas S. contains none S. contains 31 % a-helical structure and 3 spatially separated domains, whereas the pancreatic enzymes have 10-20% a-helical structure and a high content of p-structures in both types, the active center is a substrate cleft. S. also have a broader substrate specificity than the pancreatic enzymes. This is a notable example of the convergent evolution of catalytic activity in two structurally completely different classes of proteins. S. is used in the structural elucidation... 

A model for pancreatic lipase has been suggested to account for the enzyme s activity on the oil/water interface (Fig. 3.17). The lipase s hydrophobic head is bound to the oil droplet by hydrophobic interactions, while the enzyme s active site aligns with and binds to the substrate molecule. The active site resembles that of serine proteinase. The splitting of the ester bond occurs with the involvement of Ser, His and Asp residues on the enzyme by a mechanism analogous to that of chymotrypsin (cf. 2.4.2.5). The dissimilarity between pancreatic lipase and serine proteinase is in the active site lipase has a leucine residue within this site in order to establish hydrophobic contact with the lipid substrate and to align it with the activity center. 

Many proteinase inhibitors have been isolated and their structures elucidated. The active center often contains a peptide bond specific for the inhibited enzyme, e. g., Lys-X or Arg-X in trypsin inhibitors and Leu-X, Phe-X or Tyr-X... 

Table 16.14. Amino add sequences in the region of the active centers of proteinase inhibitors...

At the center of the apoptotic process lies a group of specialized cysteine-containing aspartate proteinases (see p. 176), known as cas-pases. These mutually activate one another, creating an enzyme cascade resembling the cascade involved in blood coagulation (see... 

Also see color figure.) Tissue factor-factor Vila complex. The three-dimensional structure of the complex of factor Vila and tissue factor (minus the transmembrane polypeptide domain of the tissue factor) in the absence of membrane surface. It is approximately 115 A in length and has a diameter of 40-50 A. Factor Vila shows its four distinct domains the Gla domain, two EGF-like domains, and the proteinase domain. Tissue factor contacts factor VHa via the interface between the two fibronectin type Ill-like domains. All four domains of factor Vila appear to be involved in the interaction between tissue factor and factor Vila. The Gla domain of factor Vila is folded very similarly to the Gla domain of prothrombin (Gla domain of prothrombin fragment 1). Activation of factor VII can be catalyzed by thrombin, factor Xa, factor Vila, and factor Xlla—all by cleavage at Arg -Ile . Secondary structures are shown in the center diagram two views of the close interactions between TF and factor Vila are shown in the two diagrams at each side. 

Mueller, C.G., Rissoan, M.C., Salinas, B., Ait-Yahia, S., Ravel, O., Bridon, J.M., Briere, F., Lebecque, S., and Liu, Y.J. (1997). Polymerase chain reaction selects a novel disintegrin proteinase from CD40-activated germinal center dendritic cells. J. Exp. Med. 786 655-663. 

Figure 25.3 shows the relationship of active site of serine hydrolases. The serine hydrolases include serine proteases, lipases, and PHB depolymerases. A common feature of the serine proteases is the presence of a specific amino acid sequence -Gly-Xl-Ser-X2-Gly-. The catalytic mechanism of these enzymes is very similar and the catalytic center consists of a triad of serine, histidine, and aspartate residues [54]. The serine from this sequence attacks the ester bond nucleophilically [55]. Lipases and PHB depolymerases also have a common amino acid sequence around the active site, -Gly-Xl-Ser-X2-Gly-. These serine hydrolases may share a similar mechanism of substrate hydrolysis [21, 56]. In terms of origin of enzymes, it would be wise to consider that the enzyme had wide substrate specificity initially, and then it started to evolve gradually for each specific substrate. In the case of polyester hydrolysis, lipases showed the widest substrate specificity among serine hydrolases for hydrolysis of various polyesters ranging from a-ester bonds to (o-ester bonds. PHB depolymerases would become more specific for microbial PHB that has / -ester bonds, though it could also hydrolyze other polyesters that have -ester and y-ester bonds. Serine proteases such as proteinase K, subtilisin, a-chymotrypsin, elastase, and trypsin hydrolyze only optically active PLLA with a-ester bonds and various proteins with a-amido bonds. 

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