Productive intermediates elastase

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-... 

Elastase was the first enzyme for which a productive enzyme-intermediate complex was observed crystallographically by subzero-temperature... 

G-CSF activates neutrophils, transforming them into cells capable of respiratory burst and release of secretory granules. It also modulates the expression of adhesion molecules on neutrophils as well as CD1 lb/CD18 and plasma elastase antigen levels. G-CSF induces proliferation of endothelial cells, phagocytic activity of neutrophils, reactive oxygen intermediate production by neutrophils and antibody-dependent cellular toxicity by neutrophils. 

Serine proteases are widely distributed and have many different functions. They are products of at least two evolutionary pathways, which originate in prokaryotes. Many of them resemble trypsin, chymotrypsin, elastase, or sub-tilisin in specificity, but serine proteases with quite different specificities have been isolated recently. A recent NMR study of a bacterial protease labelled with at carbon 2 of its single imidazole groups implicates a buried side chain of aspartic acid as the ultimate base for proton transfers in catalysis and eliminates a charge separation from reaction schemes for catalysis. Much of the catalytic effectiveness of serine proteases can be attributed to substrate binding, but the interactions which yield a Michaelis complex are supplemented by others which stabilize intermediates on the reaction pathway. 

Nitrogen heterocycles can be accessed by the same reductive cyclization strategy [75]. Modest yields of the desired products are obtained in these reactions (Eq. 64), which serve as intermediates in the synthesis of human neutrophil elastase inhibitors. 

A more clinically advanced inhibitor, ICI-200880 (40), now in Phase-I trials, has been designed [82] by consideration of the transition state developed during the production of the acyl-enzyme intermediate. Compound (40) is a slow binding competitive HNE inhibitor (K = 0.5 nM) which protects in the hamster model of elastase-induced emphysema for up to 48 h following administration by aerosol [83], The trifluoromethylketone (41) is the most... 

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