Presteady-state kinetic study of the elementary processes in the chymotrypsin-catalyzed hydrolysis of specific ester substrate. Rate-limiting association process due to the secondary binding.

摘要: Abstract Presteady-state kinetic studies of α-chymotrypsin-catalyzed hydrolysis a specific chromophoric substrate, N-(2-furyl)acryloyl- l -tryptophan methyl ester, were performed by using stopped-flow apparatus both under [E]0 ⪢ [S]0 and conditions in the pH range 5–9, at 25 °C. The results accounted for terms three-step mechanism involving enzyme-substrate complex (E · S) acylated enzyme (ES′); no other intermediate was observed. This substrate shown to react very efficiently, i.e., maximum second-order acylation rate constant ( k 2 K s ′ ) max = 4.2 × 10 7 M −1 . limiting values Ks′ (dissociation E S), K2 (acylation rate) k3 (deacylation obtained from profiles these parameters be 0.6 ± 0.2 10−5 m, 360 15 s−1 29.3 0.8 s−1, respectively. Likewise small observed Ki N-(2-furyl)-acryloyl- d ester Km amide. strong affinities may due intense interaction β-(2-furyl)acryloyl group with secondary binding site enzyme. led value lower than unity, rate-limiting process association, even relatively low k2 this compared those proposed labile p-nitrophenyl esters.