No abstract

No abstract

Either tris(p-mtrophenyl) phosphate or bis(p-nitrophenyl) carbonate reacts rapidly at the active site of «-chymotrypsin to release a stoichiometric amount of p-nitrophenol. The resulting phosphorylated or acylated enzyme then releases a second mole of p-nitrophenol in a process which exhibits first-order kinetics. The pH dependence of the kinetics of this second reaction, called "aging," reveals the participation of an ionizable group with a pATa near 7. This implicates the imidazole of histidine-57, in analogy to deacylation reactions. Added methanol does not appreciably accelerate phosphorylated enzyme aging reactions, but markedly does so with carbonate enzyme. Both reactions exhibit kinetic isotope effects, kulko, of 2.3-2.4, however. Phosphorylated enzyme incubated in 5 M aqueous methanol is observed to undergo a second "aging" reaction, releasing the third mole of p-nitrophenol, after which it can be reactivated by strong nucleophiles, contrary to its behavior upon incubation in the absence of methanol. Surprisingly, aged carbonate enzyme is inactive immediately after completion of the aging reaction but spontaneously reactivates thereafter in a much slower reaction step for which pH-rate studies indicate the participation of His-57. These observations suggest that carbonate ester acts as a bifunctional reagent in the active site of «-chymotrypsin. The most probable mechanisms for the chymotrypsin-catalyzed aging reactions of these esters involve nucleophilic participation of the His-57 side chain with the phosphate compound, but a general base role for this group with the carbonate ester. Labile phosphate esters, as well as esters of carboxylic > and carbonic acids, react readily and specifically

No abstract

No abstract