Elastase substrate specificity

A straightforward approach is to hunt for short polypeptides that meet the specificity requirement of an enzyme but which, because of peculiarities of the sequence, are acted upon very slowly. Such a peptide may contain unusual or chemically modified amino acids. For example, the peptide Thr-Pro-nVal-NMeLeu-Tyr-Thr (nVal=norvaline NMeLeu = N-methylleucine) is a very slow elastase substrate whose binding can be studied by X-ray diffraction and NMR spectroscopy.6 Thiol proteases are inhibited by succinyl-Gln-Val-Val-Ala-Ala-p-nitroanilide, which includes a sequence common to a number of naturally occurring peptide inhibitors called cystatins.f They are found in various animal tissues where they inhibit cysteine proteases. 

The substrate specificity of an enzyme is determined by the properties and spatial arrangement of the amino acid residues forming the active site. The serine proteases trypsin, chymotrypsin and elastase cleave peptide bonds in protein substrates on the carboxyl side of positively charged, aromatic and small side-chain amino acid residues, respectively, due to complementary residues in their active sites. 

The properties and spatial arrangement of the amino acid residues forming the active site of an enzyme will determine which molecules can bind and be substrates for that enzyme. Substrate specificity is often determined by changes in relatively few amino acids in the active site. This is clearly seen in the three digestive enzymes trypsin, chymotrypsin and elastase (see Topic C5). These three enzymes belong to a family of enzymes called the serine proteases - serine because they have a serine residue in the active site that is critically involved in catalysis and proteases because they catalyze the hydrolysis of peptide bonds in proteins. The three enzymes cleave peptide bonds in protein substrates on the carboxyl side of certain amino acid residues. 

W. Bode, E. Meyer, and J. C. POwen. Human leucocyte and pancreatic elastase X-ray structures, mechanism, substrate specificity, and mechanism-based inhibitors. Biochemistry 28 1951 (1989). 

Trypsin, chymotrypsin, and elastase are three of the most important protein-digesting enzymes secreted by the pancreas. Despite their similarities they have different substrate specificity, that is, they cleave different peptide bonds during protein digestion. 

Within each protease family, individual members will differ in their substrate specificity. Most proteases have extended substrate binding sites and will bind to and recognize several amino acid residues of a polypeptide substrate (see Figure 2). Usually one of these will be the primary binding site. For example, in the serine proteases chymotrypsin, trypsin, and elastase, the primary substrate binding site is the Si subsite... 

Older methods of classifying proteases by their substrate specificity are of no assistance with regard to the design of inhibitors. There are elastases in both the serine protease and metalloprotease family (see... 

The reaction of human leukocyte elastase has been studied with a number of azapeptide p-nitrophenyl esters and some of the results are listed in Table VII. All of the azapeptides acylate elastase except Ac-Ala-Aphe-ONp, which reacts very slowly. However this azapeptide will react with cathepsin G as expected from the differing substrate specificity of the two enzymes. The kinetics of the reaction are described in detail elsewhere (36), but with most of the inhibitors, kCSLt is equal to the deacylation (or reactivation) rate of the acylated enzyme. Azapep-... 

Trypsin cleaves a peptide bond on the C-terminal side of a basic residue such as arginine (Arg) or lysine (Lys) whereas chymotrypsin cleaves on the C-terminal side of the hydrophobic residues phenylalanine (Phe), tryptophan (Trp) or tyrosine (Tyr). Elastase cleaves on the C-terminal side of small amino acids such as alanine (Ala) and glycine (Gly). A large number of serine PI proteins have been isolated from plants (Table 13.4) and the substrate specificity of the target proteases corresponds with the inhibitory amino acid sequences (P2-P1-PT-P2 ) of the PI proteins. Thus, the double-headed trypsin- and chymotrypsin-inhibitory Bowman-Birk PI protein 1 (BBI-1) from soybean (Glycine BBI-1, Table 13.5G) has a Pl-PT sequence of Lys—Ser at the trypsin inhibitory domain I site and a PI PI sequence of Leu-Ser at the chymotrypsin inhibitory domain II site. 

The Gommission on Biochemical Nomenclature assigns enzyme numbers to 18 serine proteases in the 1972 edition of Enzyme Nomenclature (14). Seven are listed as having a trypsin-like specificity, i,e, their specific substrates have a positively charged lysine or arginine residue at Pi. Three are listed as having a chymotrypsin-like specificity, i.e., their specific substrates have residues of tryptophan, tyrosine, phenylalanine, or leucine at Pi, i,e, residues with bulky, hydrophobic side chains. Two enzymes have elastase-like specificities. They prefer a residue with... 

Many other proteins have subsequently been found to contain catalytic triads similar to that discovered in chymotrypsin. Some, such as trypsin and elastase, are obvious homologs of chymotrypsin. The sequences of these proteins are approximately 40% identical with that of chymotrypsin, and their overall structures are nearly the same (Figure 9.12). These proteins operate by mechanisms identical with that of chymotrypsin. However, they have very different substrate specificities. Trypsin cleaves at the peptide bond after residues with long, positively charged side chains—namely, arginine and lysine—whereas elastase cleaves at the peptide bond after amino acids with small side chains—such as alanine and serine. Comparison of the Sj pockets of these enzymes reveals the basis of the specificity. 

An aldehyde inhibitor. Elastase is specifically inhibited by an aldehyde derivative of one of its substrates ... 

Elastase is the name given to proteinases that possess the ability to hydrolyze mature cross-linked elastin [18]. Elastin is an insoluble structural protein responsible for the elastic properties of the lung, skin, and arteries and is quite resistant to most proteinases. Elastin is high in hydrophobic amino acid residues such as valine, alanine, glycine, and proline [19]. Insoluble elastin fibers contain cross-links usually between four lysine residues, which form a unique cyclic product, desmosine. The presence of soluble desmosine cross-links in plasma can be used as a measure of elastin breakdown. Of all the elastases in humans, neutrophil elastase has received the most attention over the years due to its broad substrate specificity and abundance within the cell. However, neutrophils and macrophages contain several proteinases (Table 1), which are capable of degrading elastin. 

Even though serine proteinases share a common mechanism of peptide bond cleavage, they differ dramatically in their primary substrate specificity, exhibiting a preference for a certain type of amino acid residue. Hydrolysis studies using natural and synthetic substrates have demonstrated that NE preferentially cleaves peptide bonds after small aliphatic amino acid residues such as valine and alanine [21]. Elastase has been shown to have a pH optimum between 8.0 and 9.0 with most protein substrates and is strongly inhibited by the plasma inhibitor ai-PI. 

Proteolytic enzymes (proteases) catalyze the hydrolysis of peptide bonds. The pancreatic serine proteases chymotrypsin, trypsin, and elastase have similar structures and mechanisms of action, but different substrate specificities. It is thought that they evolved from a common ancestral protease. 

In the recent studies, the enzyme shows that the overall polypeptide fold of chymotrypsin-like serine protease possesses essential SI specificity determinants characteristic of elastase using the multiple isomorphous replacement (MIR) method and refined to 2.3 A resolution Fig. (5). Structure-based inhibitor modeling demonstrated that EFEa s SI specificity pocket is preferable for elastase-specific small hydrophobic PI residues, while its accommodation of long and/or bulky PI residues is also feasible if enhanced binding of the substrate and induced fit of the SI pocket are achieved [Fig. (6) shows the active sites of serine protease]. EFEa is thereby endowed with relatively broad substrate specificity, including the dual fibrinolysis. This structure is the first report of an earthworm fibrinolytic enzyme component, a serine protease originated from annelid worm [17]. 

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. 

Serine proteases such as proteinase K, subtilisin, a-chymotrypsin, and elastase can hydrolyze PLLA endogenously. Their substrate specificities are relatively wide among proteins that are composed of L-amino acid unit and a-amido bonds, and specific as well, for L-lactic acid unit of PLLA (PLLA, PDLA, and PDLLA) that have a-ester bond. Serine proteases hydrolyze PLLA homopolymer faster than copolymers with low such as PDLLA, poly(lactide-co-glycolide). 

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