X-RAY CRYSTAL-STRUCTURE OF A DIPEPTIDE-CHYMOTRYPSIN COMPLEX IN AN INHIBITORY INTERACTION

The dipeptide D-leucyl-L-phenylalanyl p-fluorobenzylamide (D-Leu-Phe-N H-BzlF) inhibits chymotrypsin strongly in a competitive manner with th e K-1 value of 0.61 mu M [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. Sec. Perkin Trans. 1, 2479 -2485]. The structure/activity studies have suggested a unique inhibit ory conformation, in which the C-terminal benzyl group fits the chymot rypsin S-1 site and the hydrophobic core constructed by the side chain s of D-Leu-Phe fits the S-2 or S-1 site. To verify this assumption, th e molecular structure of the complex between the dipeptide and gamma-c hymotrypsin has been determined crystallographically. gamma-Chymotryps in itself was first crystallized and refined at 1.6-Angstrom resolutio n. The refined structure was virtually identical to the conformation r eported and the electron density at the active site was interpreted as a pentapeptide Thr-Pro-Gly-Val-Tyr derived from autolysis of the enzy me (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 ha s been refined at 1.8-Angstrom resolution to a crystallographic R-fact or 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 0.1 3 Angstrom for all the Ca carbons. Two inhibitor molecules were assign ed 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 c hains of D-Leu-Phe are spatially proximal. In the active site where th e binding of dipeptide was judged to be a direct cause of inhibition, C-terminal p-fluorobenzylamide group of the dipeptide, NH-BzlE was fou nd in the S-1 hydrophobic pocket. At the bottom of this pocket, the p- fluorine atom hydrogen bonded with a water molecule, probably to enhan ce the inhibitory activity. The stereospecific interaction of R and S isomers of the dipeptide with C-terminal NH-CH(CH3)-C6H5 was well exp lained by the space available for methyl replacement in the complex. T he hydrophobic core constructed by side chains of D-Leu-Phe was found at the bread S-2 site. Interestingly, a novel interaction was found be tween 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 Angstrom.