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J. Biochem, 1976, Vol. 80, No. 2 239-251
© 1976 Japanese Biochemical Society

research-article

Kinetic Study of {alpha}-Chymotrypsin Catalysis with Regard to the Interaction between the Specificity-determining Site and the Aromatic Side Chain of Substrates1,2

Motonori OHNO, Shin-ichi SATO3, Yuji KARASAKI and Sadaji TSUKAMOTO

Laboratory of Enzyme Chemistry, Faculty of Science, Kyushu University Higashi-ku, Fukuoka, Fukuoka 812

In order to investigate how changes in the structures of side-chain aromatic groups of specific substrates influence binding and kinetic specificity in {alpha}-chymotrypsin [EC 3.4.21. 1]-catalyzed reactions, a number of nucleus-substituted derivatives of the specific ester substrates were prepared and steady-state kinetic studies were carried out at pII 6.5 and 7.8.

Ac-Trp(NCps)-OMe was hydrolyzed more readily at low substrate concentration than Ac-Trp-OMe due to its smaller Km(app) value, suggesting that the bulky 2-nitro-4-carboxyphenylsulfenyl moiety interacts with outer residues rather than with those in the hydrophobic pocket and that this interaction increases the binding specificity. Inhibition experiments using the corresponding carboxylate and analogous inhibitors, however, showed that the carboxy group at the para position of the phenyl nucleus of the substituent sterically hinders association with the active site of {alpha}chymotrypsin at pH 7.8 but not at pH 6.5. The kcat values of Ac-Trp(CHO)-OMe, Ac-Tyr(3-NO2) OMe, and Ac-m-Tyr-OMc were much higher than those of the corresponding specific substrates, indicating that derivatives with a substituent as large as a formyl, nitro or hydroxyl group at the {varepsilon}-position are stereochemically favorable to the catalytic process. Remarkable increases in Km(app) were also observed. The individual parameters for Ac-Dopa-OMe, however, were comparable to those for Ac-Tyr-OMe.

1 This study was supported in part by a Scientific Research Grant from the Ministry of Education, Science and Culture, of Japan.

2 A preliminary account of this work has been published (l).

3 Present address: Research Institute of Biology, Yoshitomi Pharmaceutical Inc., Yoshitomi, Chikujo-gun, Fukuoka 871.


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