1oxg
From Proteopedia
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Crystal structure of a complex formed between organic solvent treated bovine alpha-chymotrypsin and its autocatalytically produced highly potent 14-residue peptide at 2.2 resolution
Overview
Chymotrypsin is a prominent member of the family of serine proteases. The, present studies demonstrate the presence of a native fragment containing, 14 residues from Ile16 to Trp29 in alpha-chymotrypsin that binds to, chymotrypsin at the active site with an exceptionally high affinity of 2.7, +/- 0.3 x 10(-11) M and thus works as a highly potent competitive, inhibitor. The commercially available alpha-chymotrypsin was processed, through a three phase partitioning system (TPP). The treated enzyme showed, considerably enhanced activity. The 14 residue fragment was produced by, autodigestion of a TPP-treated alpha-chymotrypsin during a long, crystallization process that lasted more than four months. The treated, enzyme was purified and kept for crystallization using vapour the, diffusion method at 295 K. Twenty milligrams of lyophilized protein were, dissolved in 1 mL of 25 mM sodium acetate buffer, pH 4.8. It was, equilibrated against the same buffer containing 1.2 M ammonium sulfate., The rectangular crystals of small dimensions of 0.24 x 0.15 x 0.10 mm(3), were obtained. The X-ray intensity data were collected at 2.2 angstroms, resolution and the structure was refined to an R-factor of 0.192. An extra, electron density was observed at the binding site of alpha-chymotrypsin, which was readily interpreted as a 14 residue fragment of, alpha-chymotrypsin corresponding to, Ile-Val-Asn-Gly-Glu-Glu-Ala-Val-Pro-Gly-Ser-Trp-Pro-Trp(16-29). The, electron density for the eight residues of the C-terminus, i.e., Ala22-Trp29, which were completely buried in the binding cleft of the, enzyme, was of excellent quality and all the side chains of these eight, residues were clearly modeled into it. However, the remaining six residues, from the N-terminus, Ile16-Glu21 were poorly defined although the backbone, density was good. There was a continuous electron density at 3.0 sigma, between the active site Ser195 Ogamma and the carbonyl carbon atom of, Trp29 of the fragment. The final refined coordinates showed a distance of, 1.35 angstroms between Ser195 Ogamma and Trp29 C indicating the presence, of a covalent linkage between the enzyme and the native fragment. This, meant that the enzyme formed an acyl intermediate with the autodigested, fragment Ile16-Trp29. In addition to the O-C covalent bond, there were, several hydrogen bonds and hydrophobic interactions between the enzyme and, the native fragment. The fragment showed a high complementarity with the, binding site of alpha-chymotrypsin and the buried part of the fragment, matched excellently with the corresponding buried part of Turkey ovomucoid, inhibitor of alpha-chymotrypsin.
About this Structure
1OXG is a Protein complex structure of sequences from Bos taurus with SO4 as ligand. Active as Chymotrypsin, with EC number 3.4.21.1 Full crystallographic information is available from OCA.
Reference
Detection of native peptides as potent inhibitors of enzymes. Crystal structure of the complex formed between treated bovine alpha-chymotrypsin and an autocatalytically produced fragment, IIe-Val-Asn-Gly-Glu-Glu-Ala-Val-Pro-Gly-Ser-Trp-Pro-Trp, at 2.2 angstroms resolution., Singh N, Jabeen T, Sharma S, Roy I, Gupta MN, Bilgrami S, Somvanshi RK, Dey S, Perbandt M, Betzel C, Srinivasan A, Singh TP, FEBS J. 2005 Jan;272(2):562-72. PMID:15654893
Page seeded by OCA on Tue Nov 20 23:16:32 2007
