X‐ray crystal structure of a dipeptide‐chymotrypsin complex in an inhibitory interaction

The dipeptide D ‐leucyl‐ L ‐phenylalanyl p ‐fluorobenzylamide ( D ‐Leu‐Phe‐NH‐BzlF) inhibits chymotrypsin strongly in a competitive manner with the K i value of 0.61 μM [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 S 1 site and the hydrophobic core constructed by the side chains of D ‐Leu‐Phe fits the S 2 or S.es.cf1.′.rb.ei1.rb site. To verify this assumption, the molecular structure of the complex between the dipeptide and γ‐chymotrypsin has been determined crystallographically. γ‐Chymotrypsin itself was first crystallized and refined at 1.6‐Å 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 γ‐chymotrypsin in a solution saturated with the dipeptide inhibitor. The crystal structure of the complex has been refined at 1.8‐Å resolution to a crystallographic R ‐factor of 18.1 %. The structure of γ‐chymotrypsin in the complex agreed fairly well with that of γ‐chymotrypsin per se with a rmsd of 0.13 Å for all the Cα 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 S 1 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(CH 3 )‐C 6 H 5 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 S 2 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 π‐π stacking interaction at a distance 3.75 Å.