Elastase folding

Chymotrypsinogen consists of a single 245-residue chain. The amino acid residues in chymotrypsin, trypsin, and elastase are usually all numbered according to their position in this zymogen. Inactive proenzymes are formed as precursors to enzymes of many different classes and are activated in a variety of ways. A part of the polypeptide chain of the proenzymes is often folded over the active site, interacting in a nonsubstrate-like fashion and blocking the site.197a... 

More recent studies on a series of peptide, thioester substrates which have a Pj-Ala residue (not illustrated) have shown that HLE prefers by 3-fold a P,-Nva to P,-Val residue [100]. Extension of the substrate studies to the S -subsites of elastase was accomplished with a series of compounds which had at P, to P4 a set of residues (including Ala, 8er or Thr at P, Ala, Pro or 8er at P2 and Pro at P3 or P4 ) designed to direct enzymatic cleavage to the P -P, bond. Using this region as a foundation, the substrates were sequentially extended into the P region. These studies revealed that the... 

Simultaneous with this work, the first synthetic aldehyde inhibitors of elastases were independently conceived by Thompson [122]. He had predicted that aldehydes, such as (6-3), would be transition-state inhibitors of serine proteinases (PPE). The finding that aldehyde (6-3) was approximately 1000-fold more potent as an inhibitor of PPE than the corresponding alcohol and that it had approximately 5000-fold higher affinity for PPE than the corresponding primary amide substrate (6-4) provided strong evidence in support of his argument. Also, as was described for aj-PI, the importance of the Ser-195 hydroxyl to the activity of these aldehydes was shown in a study wherein the dissociation constant between (6-3) and anhydro-PPE increased 5000-fold as compared to its potency as an inhibitor of PPE [123]. 

Recombinant a,-PI produced in yeast has also been clinically evaluated. This material is essentially identical to a -PI purified from human serum with respect to its protein sequence and its ability to inhibit neutrophil elastase however, it contains no carbohydrates. As such, one would suspect that the elimination rate following intravenous administration would be similar to that described for desialylated a -PI as described above [225]. In monkeys r-a,-PI disappeared from circulation with a half-life of 69 3 min. compared to a half-life of 2.4 0.2 days for a,-PI isolated from human serum [230], Rapid elimination following parenteral administration does not, however, preclude aerosol administration of the recombinant material. Aerosol administration of yeast-produced r-a,-PI, in doses ranging from 10 to 200 mg, resulted in a dose-dependent elevation of both antigenic recombinant a,-PI and EIC of BALF from apPI deficient individuals. Four hours after administration of a 200 mg dose of recombinant aj-PI, EIC was increased 40-fold over pre-treatment levels. Twenty-four hours post-dose, levels had declined to a 5-fold elevation relative to base-line levels. Modest increases of serum a,-PI levels were observed 24 h post-inhalation of the 200 mg dose [231], In this study, no adverse clinical reactions or evidence of sensitization to recombinant a,-PI were reported. 

Incubation in solutions of low denaturant concentration can sometimes cause inactivation. Formation of an incorrectly folded form of human carbonic anhydrase in 1.7M GuHCl which subsequently aggregates has been documented (77). Other proteins have also been observed to improperly refold at relatively low concentrations of denaturant These proteins include 6-galactosidase, tryptophanase, and elastase (6). 

Comparative studies on homologous proteins show a high conservation of the structural fold and constellation of amino acid side chains at the active site. The best example is that of the serine proteinase family of trypsin, chymotrypsin, elastase and protease A from Streptomyces griseus (see, for example, reference 97) (Fig. 10). 

Activated neutrophils release not only elastase and PR3, but also superoxide anions (05) and myeloperoxidase. These oxidants are able to react with the residue Met358 present at the active-site of ai-PI [99], which is then converted into methionine sulfoxide [100]. It has been shown that the kon of ai-PI chemically oxidized by N-chlorosucdnimide is 2000-fold lower than that of the native inhibitor [101], which means that 2000-fold more substrate breakdown will take place during the time of inhibition by the oxidized species. More recently it has been shown that this methionine residue can be oxidized in vivo where oxidants are released by alveolar macrophages [102,103] and by activated neutrophils [104]. The presence of the oxidative-sensitive methionine residue at the reactive... 

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]. 

As shown in Fig. 12.1, a 6300-fold increase in potency is observed when the hydroxylic hydrogen is replaced by a diethyl-methyl side chain. A comparable increase in potency is observed in a series of 1-methyl-1,2,3,4-tetrahydro-pyridyl-pyrazines described by Ward et al. exhibiting Mj muscarinic agonists. In changing from O-methyl to O-butyl, the aflSnity for the Mj receptor varies from 850 nM to 17 nM. Another example is found in a series of 2-pyrone-derived elastase inhibitors. ... 

Although only 24% of the residues are invariant between trypsin, chymo-trypsin, and elastase, the trypsin analysis shows that this enzyme has similar polypeptide folding, a-carbon positions, and active-site residues. Changes in the trypsin sequence from that of chymotrypsin are often compensated by further changes which allow the main-chain folding to be preserved. For instance, Met-104 of trypsin replaces a threonine in chymotrypsin, and the methionine side-chain occupies the position of Val-60 side-chain in chymotrypsin. 

Neutrophils also contain several cationic serine proteases (5). The enzyme, elastase, is particularly abundant The amount of elastase contained in the granules of a single neutrophil is in excess of one picogram (4) Several groups have used specific inhibitors as well as antisera to demonstrate that elastase or an elastase-like protease is abundant in chronic wound fluids (13,22-24). Mean levels of elastase activity is approximately 12-fold higher in fluids of chronic dermal ulcers compared to surgical... 

Comparison (or alignment) of amino acid sequences, also called homology search, often provides first-hand information on such conserved structural features and enables one to classify enzymes into families and predict the possible function of a new enzyme (86). A family of enzymes usually folds into similar 3-D structures, at least at the active site region. A typical example is the serine protease family whose members—trypsin, chymotrypsin, elastase, and subtilisin—commonly contain three active-site residues, Asp/His/Ser, which are known as the catalytic triad or charge relay system. Another example is the conserved features of catalytic domains of the highly diverse protein kinase family. In this kinase family, the ATP-binding (or phosphate-anchoring) sites present a consensus sequence motif of Gly-X-Gly-X-X-Gly (67,87). 

The interactions over a single sheet involve a folding through a right-handed supertwist which can retain interchain hydrogen bonds and the right-handed twist of each part of the pleated sheet. Such an arrangement is shown in Fig. 2.23 for the first domain of elastase. 

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