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 c onsidered axiomatic chat catalytic competence of these enzymes miners the generation of the ubiquitous catalytic site imidazolium-thiolate i on pair. We here report definitive evidence from kinetic studies suppo rted by electrostatic potential calculations, however, that at least f or some of these enzymes the ion pair state which provides the nucleop hilic and acid-base chemistry is essentially fully developed at low pH where the enzymes are inactive. Catalytic competence requires an addi tional protonic dissociation with a common pK(a) value close to 4 poss ibly from the Glu50 cluster to control ion pair geometry. The pH depen dence of the second-order rate constant (k) for the reactions of the c atalytic 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 i onizations, and all of these pK(a) values are compared with those obse rved in the pH dependence of k(cat)/K-m for the catalyzed hydrolysis o f N-acetylphenylalanylglycine 4-nitroanilide. The discrepancy between the pK value for ion pair formation and the common pK(a) value close t o 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) bu t exists also for papain (pK(a) 3.32 +/- 0.01).