J. Biochem, 1977, Vol. 81, No. 5 1273-1283
© 1977 Japanese Biochemical Society
research-article |
A Conformational Change in the Benzeneboronic Acid-
-Chymotrypsin Complex
Evidence for a Conformational Change in a Tetrahedral Adduct of 
-Chymotrypsin
Department of Physics, Faculty of Science, Kyoto University Sakyo-ku, Kyoto, Kyoto, 606
The interaction of -chymotrypsin [EC 3.4.21.1
[EC]
] with a transition state analog inhibitor, benzeneboronic acid (BBA), was investigated by the temperature-jump method using pH indicator. The relaxation signal due to the BBA-enzyme interaction was found to be independent of the BBA concentration in the concentration range examined, in contrast to the BBA-subtilisin and BBA-chymotrypsinogen systems, in which the relaxation time was dependent on the BBA concentration.
Inhibited enzymes with a tetrahedral adduct at Ser 195 were found to exhibit relaxation signals due to a unimolecular isomerization similar to that of the BBA-enzyme system even in the absence of BBA, while those with a trigonal adduct did not except for the carbamylated eazyme. The relaxation signal in DIP--chymotrypsin disappeared upon the photooxidation of His 57. The DIP-zymogen exhibited no relaxation signal under similar conditions, under which relaxation could be detected for DIP--chymotrypsin, but a relaxation signal appeared on activation by trypsin.
These observations indicate that the tetrahedral adduct at Ser 195 of -chymotrypsin has at least two different conformations. His 57 and perhaps the oxyanion hole are involved in the interaction in the tetrahedral adduct of the enzyme. Possible participation of the conformational change in the -chymotrypsin-catalyzed reaction is discussed.
1Present address: Department of Biology, Faculty of Science, Kyushu University, Higashi-ku, Fukuoka, Fukuoka 812.