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題 名 | Kinetic Study of S. griseus Aminopeptidase by Stopped-flow Fluorescence Energy Transfer |
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作 者 | 游憲政; 林萬寅; | 書刊名 | Journal of the Chinese Chemical Society |
卷 期 | 45:1 1998.02[民87.02] |
頁 次 | 頁17-22 |
分類號 | 340 |
關鍵詞 | Kinetic study; S griseus aminopeptidase; Flourescence energy transfer; |
語 文 | 英文(English) |
英文摘要 | 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 ℃ and pH8.0 are:k=(1.6 ± 0.1) × 10 �� Ms,k=0.089 ± 0.001 s, and k=0.58 ± 0.01 s. 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 ± 0.7, 35 ± 5, and 21.2 ± 0.8 kcal mol for k, k and k ��, 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. 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. |
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