Induced activation in the deacylation step of tryptic hydrolysis. An application of "inverse substrates" to mechanistic studies of the enzyme.

Trypsin [EC 3.4.21.4]-catalyzed hydrolysis of "inverse substrates" was investigated kinetically. "Inverse substrates" for trypsin are specific substrates in which the arrangement of the site-specific group is reversed compared to that of the normal substrate, e.g., a cationic center is included in the leaving group instead of being in the acyl moiety (Tanizawa, K., Kasaba, Y., & Kanaoka, Y. (1977) J. Am. Chem. Soc. 99, 4485-4488). Acyl enzyme intermediates formed specifically from these substrates are advantageous for the mechanistic analysis of trypsin action, since the cationic group liberated from the acyl moiety can no longer exhibit specific interaction with the enzyme binding site in the subsequent deacylation stage. Remarkable rate acceleration at the deacylation step was observed on adding amidinium or ammonium compounds. The effects of the size of the acyl moiety and the charged molecule on the acceleration were examined. Latent properties of 1-butylamine as an activator were found in the present study. Based on these observations, it is suggested that a cationic molecule which can be well accommodated together with the acyl group within the active center cleft causes rate enhancement, with associated conformational changes.