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

A. Kashima, Y. Inoue, S. Sugio, I. Maeda, Takeru Nose, Y. Shimohigashi

Research output: Contribution to journalArticle

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Abstract

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. and Ohno, M. (1996) J. Chem. Sec. 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, site and the hydrophobic core constructed by the side chains of D-Leu-Phe fits the S1 or S'1 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 Ca 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 π - π stacking interaction at a distance 3.75 Å.

Original languageEnglish
Pages (from-to)12-23
Number of pages12
JournalEuropean Journal of Biochemistry
Volume255
Issue number1
DOIs
Publication statusPublished - Jul 1 1998

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Dipeptides
Chymotrypsin
Crystal structure
X-Rays
X rays
leucyl-phenylalanine
Conformations
Catalytic Domain
Molecules
R388
Autolysis
Fluorine
Enzymes
Molecular Structure
Isomers
Molecular structure
Carrier concentration
Hydrogen
Carbon
Binding Sites

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

X-ray crystal structure of a dipeptide-chymotrypsin complex in an inhibitory interaction. / Kashima, A.; Inoue, Y.; Sugio, S.; Maeda, I.; Nose, Takeru; Shimohigashi, Y.

In: European Journal of Biochemistry, Vol. 255, No. 1, 01.07.1998, p. 12-23.

Research output: Contribution to journalArticle

Kashima, A. ; Inoue, Y. ; Sugio, S. ; Maeda, I. ; Nose, Takeru ; Shimohigashi, Y. / X-ray crystal structure of a dipeptide-chymotrypsin complex in an inhibitory interaction. In: European Journal of Biochemistry. 1998 ; Vol. 255, No. 1. pp. 12-23.
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abstract = "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. and Ohno, M. (1996) J. Chem. Sec. 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, site and the hydrophobic core constructed by the side chains of D-Leu-Phe fits the S1 or S'1 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-{\AA} 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-{\AA} 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 {\AA} for all the Ca 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 π - π stacking interaction at a distance 3.75 {\AA}.",
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T1 - X-ray crystal structure of a dipeptide-chymotrypsin complex in an inhibitory interaction

AU - Kashima, A.

AU - Inoue, Y.

AU - Sugio, S.

AU - Maeda, I.

AU - Nose, Takeru

AU - Shimohigashi, Y.

PY - 1998/7/1

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N2 - 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. and Ohno, M. (1996) J. Chem. Sec. 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, site and the hydrophobic core constructed by the side chains of D-Leu-Phe fits the S1 or S'1 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 Ca 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 π - π stacking interaction at a distance 3.75 Å.

AB - 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. and Ohno, M. (1996) J. Chem. Sec. 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, site and the hydrophobic core constructed by the side chains of D-Leu-Phe fits the S1 or S'1 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 Ca 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 π - π stacking interaction at a distance 3.75 Å.

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