Serine proteinase specificity

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.

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. 

The serine proteinases all have the same substrate, namely, polypeptide chains of proteins. However, different members of the family preferentially cleave polypeptide chains at sites adjacent to different amino acid residues. The structural basis for this preference lies in the side chains that line the substrate specificity pocket in the different enzymes. 

Subtilisins are a group of serine proteinases that are produced by different species of bacilli. These enzymes are of considerable commercial interest because they are added to the detergents in washing powder to facilitate removal of proteinaceous stains. Numerous attempts have therefore recently been made to change by protein engineering such properties of the subtilisin molecule as its thermal stability, pH optimum, and specificity. In fact, in 1988 subtilisin mutants were the subject of the first US patent granted for an engineered protein. 

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. 

How do the mutations identified by phage display improve binding specificity There is as yet no direct stmctural information on the phage-selected inhibitors however they can be modeled using data from the crystal structures of other Kunitz domains bound to serine proteinases. These studies lead to the conclusion that the mutations identified by phage display improve binding specificity by maximizing complementarity between the... 

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). 

Serine Proteinases. This group of proteinases is the best known because they are more numerous and better characterized than the other three groups. These proteinases are found in virtually all organisms, indicative of their importance and wide ranging proteolytic capabilities. They demonstrate broad substrate specificities with the sites (amino terminal to the scissile bond) generally being more important in enzyme interaction. 

Bivalirudin directly inhibits thrombin by specifically binding both to the catalytic site and to the anion-binding exosite of circulating and clot-bound thrombin. Thrombin is a serine proteinase that plays a central... 

The proteolytic enzymes are classified into endopeptidases and exopeptidases, according to their site of attack in the substrate molecule. The endopeptidases or proteinases cleave peptide bonds inside peptide chains. They recognize and bind to short sections of the substrate s sequence, and then hydrolyze bonds between particular amino acid residues in a relatively specific way (see p. 94). The proteinases are classified according to their reaction mechanism. In serine proteinases, for example (see C), a serine residue in the enzyme is important for catalysis, while in cysteine proteinases, it is a cysteine residue, and so on. 

Trypsin, chymotrypsin, and elastase are en-dopeptidases that belong to the group of serine proteinases (see p. 176). Trypsin hydrolyzes specific peptide bonds on the C side of the basic amino acids Arg and Lys, while chymotrypsin prefers peptide bonds of the apolar amino acids Tyr, Trp, Phe, and Leu (see p. 94). 

Glutamyl endopeptidase [EC 3.4.21.19] (also known as staphylococcal serine proteinase, V8 proteinase, protease V8, and endoproteinase Glu-C), a member of the peptidase family S2B, catalyzes the hydrolysis of Asp-Xaa and Glu-Xaa peptide bonds. In appropriate buffers, the specificity of the bond cleavage is restricted to Glu-Xaa. Peptide bonds involving bulky side chains of hydrophobic aminoacyl residues are hydrolyzed at a lower rate. 

Plasmin is a serine proteinase (inhibited by diisopropylfluorophosphate, phenylmethyl sulphonyl fluoride and trypsin inhibitor) with a high specificity for peptide bonds to which lysine or arginine supplies the carboxyl group. Its molecular weight is about 81 Da and its structure contains five intramolecular disulphide-linked loops (kringles) which are essential for its activity. 

Microbial proteinases can be classified by mechanism of action. Hartley (1960) divided them into four groups serine proteinases, thio proteinases, metalloproteinases, and acid proteinases. Morihara (1974) classified enzymes within these groups according to substrate specificity. Enzymes which split peptide substrates at the carboxyl side of specific amino acids are called carboxyendopeptidases, and those which split peptide substrates at the amino side of specific amino acids are called aminoendopeptidases. Acid proteinases, such as rennin and pepsin, split either side of specific aromatic or hydrophobic amino acid residues. The action of proteolytic enzymes on milk proteins has been reviewed by Visser (1981). 

Hansson L, Stromqvist M, Backman A, Wallbrandt P, Carlstein A, Egelrud T. Cloning, expression, and characterization of stratum corneum chymotryptic enzyme. A skin-specific human serine proteinase. J Biol Chem 1994 269 19420-19426. 

Christensson A, Laurell CB, Lilja H. Enzymatic activity of prostate-specific antigen and its reactions with extracellular serine proteinase inhibitors. Eur J Biochem. 1990 194 755-763. 

Aorsin from Aspergillus Oryzae, a Novel Serine Proteinase with Trypsinogen Activating Specificity... 

The catalytic mechanism of serine proteinases. Two protons are shown in flight at each reaction step, as is probably the case with specific substrates non-specific substrates appear to react with the operation of only one-proton catalysis (Elrod et al., 1980)... 

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). 

---