4-Chloro-7-nitrobenzo-2-oxa-1,3-diazole as a reactivity probe for the investigation of the thiol proteinases. evidence that ficin and bromelain may lack carboxyl groups conformationally equivalent to that of aspartic acid-158 of papain

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Abstract
4-Chloro-7-nitrobenzo-2-oxa-1,3-diazole (Nbd chloride) was used as a reactivity probe to characterize the active centers of papain (EC 3.4.22.2), ficin (EC 3.4.22.3) and bromelain (EC 3.4.22.4). In the pH range 0-8 Nbd chloride probably exists mainly as a monocation, possibly with the proton located on N-1 of the oxadiazole ring. Spectroscopic evidence is presented for the intermediacy of Meisenheimer-type adducts in the reaction of Nbd chloride with nucleophiles. The pH-dependence of the 2nd-order rate constants (k) of the reactions of the 3 enzymes with Nbd chloride was determined at 25.degree. C, I = 0.1 mol/l in 6.7% (vol/vol) ethanol in the pH range 2.5-5, where, at least for papain and ficin, the reactions occur specifically with their active-center thiol groups. The pH-k profile for the papain reaction is bell-shaped (pKaI = 3.24, pKaII = 3.44 and ~k = 86 M-1 .cntdot. s-1), whereas that for ficin is sigmoidal (pKa = 3.6, ~k = 0.36 M-1 .cntdot. s-1), the rate increasing with increasing pH. The profile for the bromelain reaction appears to resemble that for the ficin reaction, but is complicated by amino-group labeling. The bell-shaped profile of papain reaction is considered to arise from the reaction of the thiolate ion of cysteine-25, maintained in acidic media by interaction with the side chain of histidine-159, with the Nbd chloride monocation H-bonded at its nitro group of the un-ionized form of the carboxyl group of aspartic acid-158. The lack of acid catalysis in the corresponding reactions of ficin and probably of bromelain suggests that these enzymes may lack carboxyl groups conformationally equivalent to that of aspartic acid-158 of papain. The possible consequences of this for the catalytic sites of these enzymes is discussed.