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The pK(a) of (Asp(158))-CO(2)H of papain (EC 3.4.22.2) was determined as 2.8 by using 4-chloro-7-nitrobenzofurazan (Nbf-Cl) as a reactivity probe targeted on the thiolate anion component of the Cys(25)/His(159) nucleophilic-acid/base motif of the catalytic site. The possibility of using Nbf-Cl for this purpose was established by modelling the papain-Nbf-Cl Meisenheimer intermediate by using QUANTA/CHARMM and performing molecular orbital calculations with MOPAC interfaced with Cerius 2. A pH-dependent stopped-flow kinetic study of the reaction of papain with Nbf-Cl established that the striking rate maximum at pH 3 results from reaction in a minor ionization state comprising (Cys(25))-S(-)/(His(159))-Im(+)H (in which Im represents imidazole) produced by protonic dissociation of (Cys(25))-SH/(His(159))-Im(+)H with pK(a) 3.3 and (Asp(158))-CO(2)H. Although the analogous intermediate in the reaction of caricain (EC 3.4.22.30) with Nbf-Cl has similar geometry, the pH-k profile (k being the second-order rate constant) lacks a rate maximum under acidic conditions. This precludes the experimental determination of the pK(a) value of (Asp(158))-CO(2)H of caricain, which was calculated to be 2.0 by solving the linearized Poisson-Boltzmann equation with the program UHBD ('University of Houston Brownian dynamics'). A value lower than 2.8 had been predicted by consideration of the hydrogen-bonded networks involving Asp(158) and its microenvironments in both enzymes. The difference between these pK(a) values (values not previously detected in reactions of either enzyme) accounts for the lack of the rate maximum in the caricain reaction and for the differences in the electronic absorption spectra of the two S-Nbf-enzymes under acidic conditions. The concept of control of cysteine proteinase activity by multiple electrostatic modulators, including (Asp(158))-CO(2)(-), which modifies traditional mechanistic views, is discussed.

biochemical journal

Ionization characteristics and chemical influences of aspartic acid residue 158 of papain and caricain determined by structure-related kinetic and computational techniques: multiple electrostatic modulators of active-centre chemistry