- 作者: 游憲政 ; 林萬寅
- 中文摘要: The stopped-flow fluorescence traces for the hydrolysis of Leu-Ala-DED catalyzed by Streptomyces griseus aminopeptidase isolated from pronase exhibits a two-phase fluorescence change with comparable rates of formation and breakdown of the ES complex. We have developed a method for the determination of the individual rate constants from the stopped-flow traces. The kinetic parameters for the enzyme- catalyzed hydrolysis of Leu-Ala-DED at 25.degree.C and pH8.0 are: k/sub 1/=(1.6.plmin.0.1)*10/sup 5/M/sup -1/ s/sup -1/, k/sub -1/=0.089.plmin.0.001s/sup -1/, and k/sub 2/=0.58.plmin.0.01s/sup -1/. For the observed stopped- flow traces, the steady state approximation is valid only within a very small region around the maximal ES concentration and a large proportion (>20%) of the substrate has already been hydrolyzed during the pre-steady state. For a very fast formation of ES complex, the steady state approximation is valid for almost the entire trace. The activation energies for each individual rate constant were determined to be 10.0.plmin.0.7, 35.plmin.5, and 21.2.plmin.0.8kcal mol/sup -1/ for k/sub 1/, k/sub -1/, and k/sub 2/, respectively. Binding of E and S to form ES was accompanied by a decrease in Gibbs free energy, whereas a dramatic increase in free energy was observed for the conversion of ES to ES/sup .neq./. The dissociation of ES to form E and S had a very large activation energy, but it was also accompanied by a large increase in entropy.
- 英文摘要: --
- 中文關鍵字: Kinetics; Aminopeptidase; Stopped-Flow; Fluorescence; Energy Transfer
- 英文關鍵字: 動力學;胺基胜□;停止-流動;螢光;能量轉移