A classical enzyme active center motif lacks catalytic competence until modulated electrostatically

The cysteine proteinase superfamily is a source of natural structural variants of value in the investigation of mechanism It has long been considered axiomatic chat catalytic competence of these enzymes miners the generation of the ubiquitous catalytic site imidazolium-thiolate ion pair. We here report definitive evidence from kinetic studies supported by electrostatic potential calculations, however, that at least for some of these enzymes the ion pair state which provides the nucleophilic and acid-base chemistry is essentially fully developed at low pH where the enzymes are inactive. Catalytic competence requires an additional protonic dissociation with a common pK(a) value close to 4 possibly from the Glu50 cluster to control ion pair geometry. The pH dependence of the second-order rate constant (k) for the reactions of the catalytic sine thiol groups with 4,4'-dipyrimidyl disulfide is shown to provide the pK(a) values for the formation and deprotonation of the (Cys)-S-/C(His)-Im(+)H ion pair state, Analogous study of the reactions with 2,2'-dipyridyl disulfide reveals other kinetically influential ionizations, and all of these pK(a) values are compared with those observed in the pH dependence of k(cat)/K-m for the catalyzed hydrolysis of N-acetylphenylalanylglycine 4-nitroanilide. The discrepancy between the pK value for ion pair formation and the common pK(a) value close to I related to generation of catalytic activity is particularly marked for ficin (pK(a) 2.49 +/- 0,02) and caricain (pK(a) 2.88 +/- 0.02) but exists also for papain (pK(a) 3.32 +/- 0.01).