Thiolate-imidazolium ion pair is not an obligatory catalytic entity of cysteine peptidases: the active site of picornain 3C.

Cysteine peptidases are thought to attack the substrate by a thiolate-imidazolium ion-pair, as demonstrated with the most extensively studied papain. Picornavirus proteinases (picornains), a different family of cysteine peptidases, are structurally related to the trypsin family of serine peptidases, whose catalytically competent histidine operates as a general base catalyst. Measuring the absorbance change upon alkylation of picornains at 250 nm, where the nondissociated thiol group has a negligible absorbance relative to the ionized form, one can test the ionization state of the catalytic cysteine. For such studies, we have prepared and used a mutated variant of the poliovirus proteinase 3C, which contains a single thiol group. The pH dependence of the molar extinction coefficient has undoubtedly shown that picornain 3C contains an ordinary thiol group rather than the usual ion-pair. Therefore, the imidazole assistance, demonstrated in alkylation reactions, is presumably general base catalysis, as found with serine peptidases. Kinetic studies on k(cat)/K(m) gave large inverse deuterium isotope effects, which may overcompensate the reverse values characteristic of the potential general base catalysis. The inverse effects is associated with the stabilization of the protein structure in heavy water.