1OXG

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

Summary for 1OXG

• Entry DOI: 10.2210/pdb1oxg/pdb
• Related: 1ACB 4CHA 6CHA
• Descriptor: Chymotrypsinogen A, SULFATE ION, ... (4 entities in total)
• Functional Keywords: autocatalysis, organic solvent treatment, inhibition, hydrolase
• Biological source: Bos taurus (bovine); More
• Cellular location: Secreted, extracellular space: P00766 P00766
• Total number of polymer chains: 2
• Total formula weight: 27803.09

Authors

Singh, N.,Jabeen, T.,Sharma, S.,Roy, I.,Gupta, M.N.,Bilgrami, S.,Singh, T.P. (deposition date: 2003-04-02, release date: 2004-05-18, Last modification date: 2024-10-30)

Primary citation

Singh, N.,Jabeen, T.,Sharma, S.,Roy, I.,Gupta, M.N.,Bilgrami, S.,Somvanshi, R.K.,Dey, S.,Perbandt, M.,Betzel, C.,Srinivasan, A.,Singh, T.P.
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.
Febs J., 272:562-572, 2005

PubMed Abstract: 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.

PubMed: 15654893
DOI: 10.1111/j.1742-4658.2004.04499.x

Experimental method

X-RAY DIFFRACTION (2.2 Å)