1AFQ

CRYSTAL STRUCTURE OF BOVINE GAMMA-CHYMOTRYPSIN COMPLEXED WITH A SYNTHETIC INHIBITOR

Summary for 1AFQ

• Entry DOI: 10.2210/pdb1afq/pdb
• Related PRD ID: PRD_000279
• Descriptor: BOVINE GAMMA-CHYMOTRYPSIN, D-leucyl-N-(4-fluorobenzyl)-L-phenylalaninamide, SULFATE ION, ... (6 entities in total)
• Functional Keywords: hydrolase-hydrolase inhibitor complex, hydrolase/hydrolase inhibitor
• Biological source: Bos taurus (cattle); More
• Cellular location: Secreted, extracellular space: P00766 P00766 P00766
• Total number of polymer chains: 3
• Total formula weight: 26058.50

Authors

Sugio, S.,Kashima, A.,Inoue, Y.,Maeda, I.,Nose, T.,Shimohigashi, Y. (deposition date: 1997-03-12, release date: 1997-09-17, Last modification date: 2024-10-30)

Primary citation

Kashima, A.,Inoue, Y.,Sugio, S.,Maeda, I.,Nose, T.,Shimohigashi, Y.
X-ray crystal structure of a dipeptide-chymotrypsin complex in an inhibitory interaction.
Eur.J.Biochem., 255:12-23, 1998

PubMed Abstract: The dipeptide D-leucyl-L-phenylalanyl p-fluorobenzylamide (D-Leu-Phe-NH-BzlF) inhibits chymotrypsin strongly in a competitive manner with the Ki value of 0.61 microM [Shimohigashi, Y., Maeda, I., Nose, T., Ikesue, K., Sakamoto, H., Ogawa, T., Ide, Y., Kawahara, M., Nezu, T., Terada, Y., Kawano, K. & Ohno, M. (1996) J. Chem. Soc. Perkin Trans. 1, 2479-2485]. The structure/activity studies have suggested a unique inhibitory conformation, in which the C-terminal benzyl group fits the chymotrypsin S1 site and the hydrophobic core constructed by the side chains of D-Leu-Phe fits the S2 or S1' site. To verify this assumption, the molecular structure of the complex between the dipeptide and gamma-chymotrypsin has been determined crystallographically. Gamma-chymotrypsin itself was first crystallized and refined at 1.6-A resolution. The refined structure was virtually identical to the conformation reported and the electron density at the active site was interpreted as a pentapeptide Thr-Pro-Gly-Val-Tyr derived from autolysis of the enzyme (residues 224-228). The chymotrypsin-dipeptide complex was obtained by soaking the crystals of gamma-chymotrypsin in a solution saturated with the dipeptide inhibitor. The crystal structure of the complex has been refined at 1.8-A resolution to a crystallographic R-factor of 18.1%. The structure of gamma-chymotrypsin in the complex agreed fairly well with that of gamma-chymotrypsin per se with a rmsd of 0.13 A for all the C alpha carbons. Two inhibitor molecules were assigned in an asymmetric unit, i.e. one in the active site and the other at the interface of two symmetry-related enzyme molecules. In both sites dipeptides adopted very similar folded conformations, in which side chains of D-Leu-Phe are spatially proximal. In the active site where the binding of dipeptide was judged to be a direct cause of inhibition, C-terminal p-fluorobenzylamide group of the dipeptide, NH-BzlF, was found in the S1 hydrophobic pocket. At the bottom of this pocket, the p-fluorine atom hydrogen bonded with a water molecule, probably to enhance the inhibitory activity. The stereospecific interaction of R and S isomers of the dipeptide with C-terminal NH-C*H(CH3)-C6H5 was well explained by the space available for methyl replacement in the complex. The hydrophobic core constructed by side chains of D-Leu-Phe was found at the broad S2 site. Interestingly, a novel interaction was found between the inhibitor Phe residue and chymotrypsin His57, the phenyl of Phe and the imidazole of His being in a pi-pi stacking interaction at a distance 3.75 A.

PubMed: 9692896
DOI: 10.1046/j.1432-1327.1998.2550012.x

Experimental method

X-RAY DIFFRACTION (1.8 Å)