Serine endopeptidase specificity

This enzyme [EC 3.4.21.62], a serine endopeptidase that evolved independently of chymotrypsin, contains no cys-teinyl residues. This enzyme catalyzes the hydrolysis of peptide bonds in proteins and has a broad specificity, with a preference for a large uncharged aminoacyl residue in the PI subsite. 

Another beneficial property of the commonly used Bacillus subtilisins is their broad substrate specificity. In contrast to other serine endopeptidases like trypsin and chymotrypsin, the subtilisins have elongated substrate binding sites that allow them to accommodate a wide variety of different peptide sequences. 

Krystek S, Stouch T, Novotny J. Affinity and specificity of serine endopeptidase-protein inhibitor interactions. Empirical free energy calculations based on X-ray crystallographic structures. J Mol Biol 1993 234 661-679. 

S. Krystek, T. Stouch, and J. Novotny, ]. Mol. Biol., 234,661 0 993). Affinity and Specificity of Serine Endopeptidase-Protein Inhibitor Interactions Empirical Free Energy Calculations Based on X-Ray Crystallographic Structures. 

The serine endopeptidases include the chymotrypsin family (EC 3.4.21.1), trypsin (EC 3.4.21.4), elastase (EC 3.4.21.37), thrombin (EC 3.4.21.5), subtilisin (EC 3.4.21.62) and a-lytic proteases (EC 3.4.21.12). The enzymes are all endopeptidases. The substrate specificities of the individual members of this group are often quite different, which is attributed to different structures of the binding pockets. 

The mechanism of hydrolysis of cysteine peptidases, in particular cysteine endopeptidases (EC 3.4.22), shows similarities and differences with that of serine peptidases [2] [3a] [55 - 59]. Cysteine peptidases also form a covalent, ac-ylated intermediate, but here the attacking nucleophile is the SH group of a cysteine residue, or, rather, the deprotonated thiolate group. Like in serine hydrolases, the imidazole ring of a histidine residue activates the nucleophile, but there is a major difference, since here proton abstraction does not appear to be concerted with nucleophilic substitution but with formation of the stable thiolate-imidazolium ion pair. Presumably as a result of this specific activation of the nucleophile, a H-bond acceptor group like Glu or Asp as found in serine hydrolases is seldom present to complete a catalytic triad. For this reason, cysteine endopeptidases are considered to possess a catalytic dyad (i.e., Cys-S plus H-His+). The active site also contains an oxyanion hole where the terminal NH2 group of a glutamine residue plays a major role. 

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. 

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. 

Trypsin, chymotrypsin, and elastase are serine proteases (see Chapter 9) that act as endopeptidases. Trypsin is the most specific of these enzymes, cleaving peptide bonds in which the carboxyl (carbonyl) group is provided by lysine or arginine (see Fig. 37.3). Chymotrypsin is less specific but favors residues that contain hydrophobic or acidic amino acids. Elastase cleaves not only elastin (for which it was named) but also other proteins at bonds in which the carboxyl group is contributed by amino acid residues with small side chains (alanine, glycine, or serine). The actions of these pancreatic endopeptidases continue the digestion of dietary proteins begun by pepsin in the stomach. 

Chymotrypsin is only one member of the family of serine proteases (enzymes that utilize an active-site serine (Ser, S) to cleave the peptide chain). Trypsin, also an endopeptidase obtained from bovine pancreas, is another member of the same family of serine (Ser, S) proteases. Interestingly, however, trypsin cleaves peptides on the carbon side of lysine (Lys, K) and arginine (Arg, R) groups. The catalytic triad discussed above for chymotrypsin also appears to apply here, while still other peptidases have other conserved units that allow them to catalyze the cleavage of peptides at specific sites. While it is not appropriate to provide an exhaustive list of peptidases that have been found to effect protein cleavage, it is important to be aware that in addition to serine (Ser, S) proteases, there are cysteine (Cys, C) proteases and aspartate (Asp, D) proteases and, in addition to endopeptidases, there are aminopeptidases that act on N-termini (N-terminal exopeptidases) and carboxy-peptidases (carboxyl-terminus exopeptidases) that act at the corresponding carboxylic acid termini. 

Some endopeptidases are very specific in cleaving peptide bonds. For example, the Staphylococcus aureus V8 endopeptidase will break only that peptide bond whose carbonyl function has been contributed by glutamic acid (in other words the peptide bond must have a glutamic acid residue lying towards the amino terminal end) bovine pancreas elastase will break only those peptide bonds whose carbonyl function is contributed by alanine, glycine, serine, or valine. 

A trypsin-related bacterial enzyme that was tested in coupling reactions is the lysine-specific serine protease I from Achromobacter lyticus. The enzyme is secreted and widely used in sequence analysis of proteins [55]. It was also used in a chemo-enzymatic route for the production of human insulin from porcine insulin. Since this endopeptidase cleaves only after Lys, it could be applied for replacing the C-terminus of the insulin B-chain from -Lys-Ala (porcine C-terminal sequence) to -Lys-Thr (human C-terminus) in a two-step or a single-step reaction. Using the B-chain as the acyl donor and Thr-OBu in DMF-ethanol mixtures as the nucleophile, a high conversion (85-90%) was obtained [9,56]. Trypsin could also be applied in this biotransformation but required higher enzyme loading. 

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