Chymotrypsin inhibition induced by side chain-side chain intramolecular CH/π interaction in D-Thr-L-Phe benzylamide

Iori Maeda, Yasuyuki Shimohigashi, Koichi Ikesue, Takeru Nose, Yuzuru Ide, Keiichi Kawano, Motonori Ohno

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Abstract

The dipeptide benzyl amide H-D-Thr-Phe-NH-CH2-C6H5 was found to inhibit chymotrypsin strongly (K(i) = 4.5 x 10-6 M) in a competitive manner. When a series of phenyl amides H-D-Thr-Phe-NH-(CH2)(n)-C6H5 (n = 0-4) were tested, inhibitory potency peaked at n = 1 (benzyl amide). Incorporation of a methyl group into the benzyl methylene resulted in formation of stereoisomers, H-D-Thr-Phe-NH-(R or S)-CH(CH3)-C6H5, with considerably different inhibitory potencies. The R-isomer was as active as the benzyl amide, while the S-isomer was about 30-fold less active than the benzyl amide. Furthermore, when a fluorine atom was introduced into the para-position of the amide-benzyl group, the resulting H-D-Thr-Phe-NH-CH2-C6H4(p-F) showed considerably enhanced inhibitory activity (about 5-fold, K(i) = 9.1 x 10-7 M). In conformational analysis by 400 mHz 1H-NMR, all dipeptides having D-Thr-Phe backbone structure showed large upheld shifts of D-Thr-βOH (shifts in ppm, 0.09-0.17), D-Thr-βCH (0.23-0.32), and D-Thr-γCH3 (0.38-0.53), indicating the presence of shielding effects from the benzene ring. In addition, NOE enhancements between the D-Thr-γCH3 and Phe-phenyl groups were evidenced by measurements of two-dimensional NOESY spectra and NOE difference spectra. These observations demonstrated the spatial proximity of these side chains, which is due to side chain-side chain CH/π interaction. All these results support the idea that the amide-benzyl group binds at the chymotrypsin S1 site, while the hydrophobic core with CH/π interaction binds at the S2 or S1' site.

Original languageEnglish
Pages (from-to)870-877
Number of pages8
JournalJournal of Biochemistry
Volume119
Issue number5
Publication statusPublished - May 1 1996

All Science Journal Classification (ASJC) codes

  • Biochemistry

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  • Cite this

    Maeda, I., Shimohigashi, Y., Ikesue, K., Nose, T., Ide, Y., Kawano, K., & Ohno, M. (1996). Chymotrypsin inhibition induced by side chain-side chain intramolecular CH/π interaction in D-Thr-L-Phe benzylamide. Journal of Biochemistry, 119(5), 870-877.