Elastase structure

To prove that any complex which formed at the low temperature was both productive and covalent, two additional experiments were carried out. First, an attempt was made to wash the substrate out of the enzyme at low temperature. The crystal was held at -55 C and substrate-free 70% methanol was flowed over it for 4 days. There was no change in the substrate-sensitive reflections, which were monitored every 8 hours during this period, and when another data set was collected at the end of the wash, it revealed the substrate still bound in the active site. However, when the crystal was allowed to warm up to - 10°C, the monitor reflections immediately began to change in intensity, back to the values they had for the native enzyme. In less than 20 hours all of them had returned to these values, and a final set of data was collected as expected, on processing it showed an empty active site and a native elastase structure. These two control experiments indicated that the structure that formed when elastase was exposed to the ester substrate was covalent, and that the covalent intermediate would undergo hydrolysis (presum-... 

Figure 4.5 Assignment of atom types for the N and O atoms in Gin sidechains can be difficult and often relies on interpreting the H-bond partners in the local environment. For the two elastase structures lela (green carbon atoms) and lelc (yellow carbon atoms), a donor and an acceptor group in the ligand form close contacts with the Gin amide group, respectively. This allows unambiguous assignment of the O and N atom types. The position and orientation of the amide plane are almost identical in the two structures only the O and N positions are switched. The figure was prepared using RasMol [147].

J. Bieth. Elastases structure, function and pathological role. In Frontiers of Matrix Biology (L. Robert, ed.). Karger, Basel, 1978, pp. 1-82. 

Magn. Reson. Spectrosc., Vol 48, No.l,(March 2006), pp 1-23, ISSN 00796565 Bieth JG. (1978). Elastases Structure, function, and pathological role. Front Matrix Biol,Vol. 6,... 

Bode, W., Papamokos, E., Musil, D. The high-resolution X-ray crystal structure of the complex formed between subtilisin Carlsberg and eglin c, an elastase inhibitor from the leech Hirudo medicinalis. Eur. J. Biochem. 166 (1987) 673-692... 

This is nicely illustrated by members of the chymotrypsin superfamily the enzymes chymotrypsin, trypsin, and elastase have very similar three-dimensional structures but different specificity. They preferentially cleave adjacent to bulky aromatic side chains, positively charged side chains, and small uncharged side chains, respectively. Three residues, numbers 189, 216, and 226, are responsible for these preferences (Figure 11.11). Residues 216... 

Shotton, D.M., Watson, H.C. Three-dimensional structure of tosyl-elastase. Nature 225 811-816, 1970. 

Trypsin, chymotrypsin, and elastase all carry out the same reaction—the cleavage of a peptide chain—and although their structures and mechanisms are quite similar, they display very different specificities. Trypsin cleaves peptides... 

Transition state theory, 46,208 Transmission factor, 42,44-46,45 Triosephosphate isomerase, 210 Trypsin, 170. See also Trypsin enzyme family active site of, 181 activity of, steric effects on, 210 potential surfaces for, 180 Ser 195-His 57 proton transfer in, 146, 147 specificity of, 171 transition state of, 226 Trypsin enzyme family, catalysis of amide hydrolysis, 170-171. See also Chymotrypsin Elastase Thrombin Trypsin Plasmin Tryptophan, structure of, 110... 

Further, Wasserman and coworkers developed a direct acylation of stabilized phosphonium ylides by carboxylic acids in presence of the EDCI/DMAP (way c). This last method allows the introduction of a-aminoacid structures into the resulting P-oxo phosphorus ylides [19-25],opening the way to the total synthesis of depsipeptide elastase inhibitors [22,24] or cyclic peptidic protease inhibitor EurystatinA [20]. 

Irreversible inhibition is probably due to the alkylation of a histidine residue.43 Chymotrypsin is selectively inactivated with no or poor inhibition of human leukocyte elastase (HLE) with a major difference the inactivation of HLE is transient.42,43 The calculated intrinsic reactivity of the coumarin derivatives, using a model of a nucleophilic reaction between the ligand and the methanol-water pair, indicates that the inhibitor potency cannot be explained solely by differences in the reactivity of the lactonic carbonyl group toward the nucleophilic attack 43 Studies on pyridyl esters of 6-(chloromethyl)-2-oxo-2//-1 -benzopyran-3-carboxylic acid (5 and 6, Fig. 11.5) and related structures having various substituents at the 6-position (7, Fig. 11.5) revealed that compounds 5 and 6 are powerful inhibitors of human leukocyte elastase and a-chymotrypsin thrombin is inhibited in some cases whereas trypsin is not inhibited.21... 

The acyl-enzyme can eliminate the 4-chlorine atom to generate this reactive intermediate that can then react with a nearby nucleophile such as His57 to give an alkylated acyl-enzyme derivative in which the inhibitor moiety is bound to the enzyme by two covalent bonds (Scheme 11.5). Inhibition is irreversible.59 The mechanism has been confirmed by X-ray structural analysis of protease-isocoumarin complexes. There is a cross-link between the inhibitor and the Serl95 and His57 residues of PPE.60 Human leukocyte elastase is also very efficiently inactivated.61... 

Doucet, C. Pochet, L. Thierry, N. Pirotte, B. Delarge, J. Reboud-Ravaux, M. 6-Substituted 2-OXO-2H-1 -benzopyran-3-carboxyI ic acid as a core structure for specific inhibitors of human leukocyte elastase. J. Med. Chem. 1999, 42, 4161—4171. 

Vijayalakshmi, J. Meyer, E. F. Kam, C.-M. Powers, J. C. Structural study of porcine pancreatic elastase complexed with 7-amino-3-(2-bromoethoxy)-4-chloroisocoumarin as a nonreactivable doubly covalent enzyme-inhibitor complex. Biochemistry 1991, 30, 2175-2183. 

Eosinophils may be increased in some patients, particularly during exacerbations. Activated inflammatory cells release a variety of mediators, most notably leukotriene B4, interleukin-8, and tumor necrosis factor-a (TNF-a). Various proteinases, such as elastase, cathepsin G, and proteinase-3, are secreted by activated neutrophils. These mediators and proteinases are capable of sustaining inflammation and damaging lung structures. 

A different strategy for measuring protease activity is based on the property of xanthene dyes to form H-type dimers (see Sect. 6.2.3) when they are in close proximity. These dimers are accompanied with a characteristic quenching of their fluorescence and, particularly for rhodamines, with a blue shift in the absorption spectrum [121, 122]. The probe D-NorFES-D designed to measure activity of elastase in HL-60 cells consists of an undecapeptide derivatized with one tetramethylrhodamine dye on each side. The sequence contains proline residues to create a bent structure and bring the two fluoro-phores in close proximity. Intact D-NorFES-D shows 90% of its fluorescence quenched plus a blue shift of the absorption spectrum. After addition of the serine protease elastase, an increase in the fluorescence and a bathochromic shift of the absorption spectrum is observed, resulting in an increase in the emission ratio [80],... 

Cathepsin G, a cationic, glycosylated protein of relative molecular mass -27 kDa, exists in four isoforms (25-29 kDa) that are identical in amino acid sequence but differ in levels of glycosylation. It is a component of azurophilic granules and present in human neutrophils at 1.5-3 jug/106 cells, but at lower levels in monocytes. cDNA has been cloned and sequenced (and the amino acid sequence predicted), and the gene has been localised to chromosome 14ql 1.2. The gene comprises five exons and four introns, a structure similar to that of the elastase gene. 

Pohl, J., Pereira, A., Martin, N. M., Spitznagel, J. K. (1990). Amino acid sequence of CAP37, a human neutrophil granule-derived antibacterial and monocyte-specific chemotactic glycoprotein structurally similar to neutrophil elastase. FEBS Lett. 272, 200-4. 

Differences between the spectra of fluorescence and phosphorescence are immediately obvious. For all tryptophans in proteins the phosphorescence spectrum, even at room temperature, is structured, while the fluorescence emission is not. (Even at low temperatures the fluorescence emission spectrum is usually not structured. The notable exceptions include a-amylase and aldolase, 26 protease, azurin 27,28 and ribonuclease 7, staphylococcal endonuclease, elastase, tobacco mosaic virus coat protein, and Drosophila alcohol dehydrogenase 12. )... 

X. C. Ding, B. F. Rasmussen, G. A. Petsko, D. Ringe, Direct Structural Observation of an Acyl-Enzyme Intermediate in the Hydrolysis of an Ester Substrate by Elastase , Biochemistry 1994, 33, 9285-9293. 

The /3-lactam structure can also react with active-serine hydrolases other than PBPs and /3-lactamases. It has been shown that appropriately substituted cephalosporins (e.g., 5.18) are potent mechanism-based inactivators of human leukocyte elastase (HLE, EC 3.4.21.37), a serine endopeptidase involved in the pathogenesis of pulmonary emphysema and other connective tissue diseases [57-60]. Subsequent work has demonstrated that substituted /3-lactams such as 5.19 or 5.20 are more stable HLE inhibitors and have improved potencies [61-63]. 

Fig. 68. Schematic backbone drawing of the elastase molecule, showing the similar 0 barrel structures of the two domains. The outside surfaces of the 0 barrels are stippled.

Ding X, Rasmussen BF, Petsko GA, Ringe D. 1994. Direct structural observation of an acyl-enzyme intermediate in the hydrolysis of an ester substrate by elastase. Biochemistry 33 9285-9293. 

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