Peptidyl thioamides as substrates and inhibitors of papain, and as probes of the kinetic significance of the oxyanion hole.

The interaction of papain with a series of amide and thioamide substrates was studied to assess the contribution of the oxyanion hole to catalysis. Amides 1a-4a (AcPheGly-NHR, where R = H, CH3, PhCH2 or p-O2NC6H4, respectively) were all hydrolyzed to AcPheGly-OH with kcat/Km values from 23-430 M-1s-1 (25 degrees C, 20% v/v MeCN in 50 mM phosphate buffer, pH 6.3). Structurally analogous thioamides 1b-3b (AcPheGlyTNHR) were not detectably hydrolyzed by papain, but 4b (AcPheGlyTNHC6H4NO2) was hydrolyzed to the thiolacid AcPheGly-SH (kcat/Km = 2125 M-1s-1). The latter was hydrolyzed further to AcPheGly-OH in a slower reaction. Thioamides 1b-3b bound to papain and inhibited the papain-catalyzed hydrolysis of Z-Gly-ONp, but the inhibition was generally less than 50% at concentrations up to 500 microM, suggesting that the binding was purely non-covalent. The inability of papain to hydrolyze 1b-3b while 1a-3a are excellent substrates suggests that the oxyanion hole plays an important role in amide hydrolysis by papain. The facile hydrolysis of thioamide 4b was attributed to decreased amide bond resonance (i.e. a more reactive ground state) caused by the strong electron-withdrawing effect of the p-nitrophenyl substituent.