Elastase crystals, activity

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-... 

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... 

R. C. Hubbard, F. Ogushi, G. A. Fells, A. M. Cantin, S. Jallat, M. Courtney, and R. G. Crystal. Oxidants spontaneously released by alveolar macrophages of cigarette smokers can inactivate the activate the active site of at-antitrypsin, rendering it ineffective as an inhibitor of neutrophil elastase. J. Clin. Invest. 0 1289 (1987). 

The design of peptidomimetic HLE inhibitors by researchers at Zeneca Pharmaceuticals23-2Z as based largely on information obtained from the X-ray crystal structures of peptidic inhibitors bound to HLE and the closely related enzyme, porcine pancreatic elastase (PPE). These two enzymes show relatively high structural homology, especially in the active site region. 

Vandlen and Tulinsky report a 3.5 A resolution comparison of the structure of a-chymotrypsin at pH 6.7 with that at pH 3.9, which shows that there has been a conformational change between these pH values. However, a comparison of the structures of native elastase at pH 5 and pH 8.S shows that, in both cases, the active-site histidine is hydrogen-bonded to Asp-102 and Ser-195 as found in a-chymotrypsin. The only difference appears to be the presence of two bound sulphates at pH S, one of which lies 6 A from the side-chains of His-57 and Ser-195. In a similar way there appears to be no conformational change in the crystals of DIP-inhibited trypsin between pH 5 and pH 9, although there are conformational chan in solution in both trypsin and trypsinogen between these values. 

---