Elastase crystal

Fig. 17. Plots of the changes in intensity with time of two high-resolution reflections from an elastase crystal as substrate is added in a flow cell. The solid curves are calculated assuming a first-order rate constant of 5.4 x 10 sec . ...

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... 

Proteins crystallized from very low salt concentrations (examples are carboxypeptidase A and elastase) can often be treated exacdy like proteins crystallized from alcohol-water mixtures. Their low solubility in water allows them to be transferred from their normal mother liquor to a distilled water solution or to a solution of low (10-20%) alcohol concentration without disorder. It is advisable to carry out this transfer at near 0 C to further decrease the protein solubility. From this stage it is trivial to add alcohol while cooling, as described above. Complications arise, however, when the salt employed as a precipitant in the native mother liquor is insoluble in alcohols. The solution to this problem is to replace the salt by ammonium acetate at equivalent or higher ionic strength. Ammonium acetate is soluble up to 1 M in pure methanol, and is very soluble in nearly all alcohol-water mixtures, even at low temperature. It therefore provides a convenient substitute for salts such as sodium sulfate or sodium phosphate. 

The presence of a covalent acyl-enzyme intermediate in the catalytic reaction of the serine proteases made this class of enzymes an attractive candidate for the initial attempt at using subzero temperatures to study an enzymatic mechanism. Elastase was chosen because it is easy to crystallize, diffracts to high resolution, has an active site which is accessible to small molecules diffusing through the crystal lattice, and is stable in high concentrations of cryoprotective solvents. The strategy used in the elastase experiment was to first determine in solution the exact conditions of temperature, organic solvent, and proton activity needed to stabilize an acyl-enzyme intermediate for sufficient time for X-ray data collection, and then to prepare the complex in the preformed, cooled crystal. Solution studies were carried out in the laboratory of Professor A. L. Fink, and were summarized in Section II,A,3. Briefly, it was shown that the chromophoric substrate -carbobenzoxy-L-alanyl-/>-nitrophenyl ester would react with elastase in both solution and in crystals in 70 30 methanol-water at pH 5.2 to form a productive covalent complex. These... 

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-... 

L.H. Takashashi, R. Radhakrishnan, R.E. Rosenfield, E.F. Meyer, D.A. Trainor, Crystal structure of the covalent complex formed by a peptidyl a,a-difluoro- -keto amide with porcine pancreatic elastase at 1.78 A resolution, J. Am. Chem. Soc. Ill (1989) 3368-3374. 

Creutzfeldt-Jakob disease 248 Crick, Francis H. C. 84, 200 Cristae of mitochondria 14 Crossing-over 18 Crosslinking 79 Crotonase. See Enoyl hydratase Crowfoot Hodgkin, Dorothy M. 84 Cruciform structure in nucleic acids 229 Crustacea 24 Cruzain 619 Cryoenzymology 469 elastase 616 Cryoprotectants 191 Crystallins 169 Crystallography 131-137 electron 131 X-ray 132-137 Crystals, liquid 392-394 Crystal systems 133 Cubic symmetry... 

The mammalian serine proteases appear to represent a classic case of divergent evolution. All were presumably derived from a common ancestral serine protease.23 Proteins derived from a common ancestor are said to be homologous. Some nonmammalian serine proteases are 20 to 50% identical in sequence with their mammalian counterparts. The crystal structure of the elastase-like protease from Streptomyces griseus has two-thirds of the residues in a conformation similar to those in the mammalian enzymes, despite having only 186 amino acids in its sequence, compared with 245 in a-chymotrypsin. The bacterial enzymes and the pancreatic ones have probably evolved from a common precursor. 

We have extended these studies to five /V-bcnzoylazolcs (imidazole, pyrazole, indole, benzimidazole and carbazole) [35] but this time centered on crystallography and solid state NMR. 9-Benzoylcarbazole (29) has a low barrier (29.7 kJ mol1) and shows spontaneous resolution (both axial enantiomers were separated by crystallization). Recently, /V-benzoylpyrazoles have been described as inhibitors of human neutrophil elastase [36],... 

H.P. Schnebli, R.C. Thompson, and R.G. Crystal. 1990. Aerosolization of recombinant SLPI to augment antineutrophil elastase protection of pulmonary epithelium. /. Appl. Physiol. 69 1843-1848. 

H. Takahashi,T. Nnkiwa, K. Yoihhnum, G. D. Quick. D. J. States, 3. Whang-PeoR. T. Knrnsen, and R. O. Crystal. Structure of the human neutrophil elastase gene. J.BioL Chem 263 14739 (1988). 

C. Vagelmcyer,R.G Hubbard, O. A. FelbrH.P.Sclinebli, R. C. Thompson, H. Fritz, and R. G. Crystal. Anti-neutiophil elastase defense of the normal human respiratory... 

Powers JC, Oleksyszyn J, Narasimhan SL, Kam C-M, Radhakrishnan REF, Meyer J (1990) Reaction of Porcine pancreatic elastase with 7-substituted 3-alkoxy-4-chloroisocoumarins design of potent inhibitors using the crystal structure of the complex formed with 4-chloro-3-ethoxy-7-guanidinoisocoumarin. Biochemistry 29 3108-3118... 

Molecular modeling studies were performed using the crystal structure of human elastase (pdb code IHNE), Sybyl molecular modeling software and a Tripos forcefield. An assumption was made that the silicon diol forms a covalent bond with the catalytic serine residue, Serl95. 

Mattos, C., Giammona, D. A., Petsko, G. A. and Ringe, D. (1995) Structural analysis of the active site of porcine pancreatic elastase based on the X-ray crystal structures of complexes with trifluoroacetyl-dipeptide-anilide inhibitors. Biochemistry, 34, 3193-3203. 

X-ray analysis of crystals of iohexol and serine proteinase (pancreaticporcine elastase) reveals that three molecules of iohexol are associated with elastase, with one close to the active site (subsite SI), the second in the vicinity of in subsites S2/S3, and the third located in a pocket at the surface of the protein. The association is a result of the affinity of iohexol directed toward the hydrophobic regions of the enzyme and supports the hypothesis of the contrast medium s potent inhibition of thrombin. Another example of the contrast medium-protein interaction is between iopamidol and fibrinogen or lysozyme... 

Applications of low temperature work in structural studies have been described in section 3(b). Application to enzyme action is best exemplified by the pioneering work of Fink and Ahmed [221] and Alber etal. [222] on elastase. JV-Carbobenzoxy-L-alanyl-p-nitrophenol ester was selected for study at — 55°C in a 70% methanol-water mixture. Kinetic studies in the presence of cryoprotectant enabled conditions for formation and stabilisation of the acyl-enzyme intermediate to be established. By monitoring changes in intensity of certain reflections as substrate flowed past the crystal at — 55°C, it was possible to show that the rate of formation of the acyl-enzyme was comparable to that obtained by monitoring p-nitrophenol release spectroscopically. The difference electron density map at 3.5 A resolution showed a peak consistent with the formation of an acyl-enzyme intermediate, but a detailed mechanistic interpretation requires higher resolution data. When the crystal was warmed to — 10°C and the data recollected, the peak in the difference synthesis disappeared, indicating that deacylation had occurred, consistent with the predictions from kinetic studies. 

M. D. Wewers, M. A. Casolaro, and R. G. Crystal. Comparison of alpha-1-antitrypsin levels and antineutrophil elastase capacity of blood and lung in a patient with the alpha-1-antitrypsin phenotype null-null before and during alpha-1-antitrypsin augmentation therapy. Am. Rev. Respir. Dis. 735 539 (1987). 

J. E. Gadek, G. A. Fells, D. G. Wright, and R. G. Crystal. Human neutrophil elastase functions as a type III collagen collagenase. Biochem. Biophys. Res. Comm. 98 1815(1980). 

M. A Casolaro, G. Fells, M. Wewers, J. E. Pierce, F. Ogushi, R. Hubbard, S. Sellers, J. Forstrom, D. Lyons, G. Kawasaki, and R. G. Crystal. Augmentation of lung antineutrophil elastase capacity with recombinant human a-1-antitrypsin. J. Appl. Physiol. 65 2015 (1987). 

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