著者

MAEDA Iori SHIMOHIGASHI Yasuyuki IKESUE Koichi NOSE Takeru IDE Yuzuru KAWANO Keiichi OHNO Motonori

出版者

The Japanese Biochemical Society

雑誌

The journal of biochemistry (ISSN:0021924X)

巻号頁・発行日

vol.119, no.5, pp.870-877, 1996-05-01

参考文献数

25

被引用文献数

5

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The dipeptide benzyl amide H-D-Thr-Phe-NH-CH₂-C₆H₅ was found to inhibit chymotrypsin strongly (K_i=4.5×10^-6M) in a competitive manner. When a series of phenyl amides H-D-Thr-Phe-NH-(CH₂)<i>_n</i>-C₆H₅ (<i>n</i>=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-(<i>R</i> or <i>S</i>)-CH(CH₃)-C₆H₅, with considerably different inhibitory potencies. The <i>R</i>-isomer was as active as the benzyl amide, while the <i>S</i>-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(<i>p</i>-F) showed considerably enhanced inhibitory activity (about 5-fold, <i>K</i>_i=9.1×10^-7M). In conformational analysis by 400MHz ¹H-NMR, all dipeptides having D-Thr-Phe backbone structure showed large upfield shifts of D-Thr-βO<i>H</i> (shifts in ppm, 0.09-0.17), D-Thr-βC<i>H</i> (0.23-0.32), and D-Thr-γC<i>H</i>₃ (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-dimen-sional 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.