Enzyme Inhibition Assays Using Fluorescence Correlation Spectroscopy: A New Algorithm for the Derivation of kcat/KM and Ki Values at Substrate Con
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- 作者：Franz-Josef Meyer-Almes and Manfred Auer
- 刊名：Biochemistry
- 出版年：2000
- 出版时间：October 31, 2000
- 年：2000
- 卷：39
- 期：43
- 页码：13261 - 13268
- 全文大小：78K
- 年卷期：v.39,no.43(October 31, 2000)
- ISSN：1520-4995
文摘
A new mathematical formalism is deduced which allows for the calculation of the kcat overKM ratio based on measurements of the enzyme kinetics with substrate concentrations much lower thanKM. The equations are also applied on the action of an inhibitor on enzyme activity yielding the bindingconstant, Ki, of an inhibitor molecule. For practical evaluation of the new theoretical approach, the enzymaticreaction of CD45 phosphatase was used as a well-characterized model system with known inhibitors fortesting the Ki value determination scheme. The kcat/KM ratio was calulated to be 4.7 × 105 M-1 s-1, theKi of the inhibitor molecule PKF52-524 was estimated to be (1-2) × 10-7 M and the association rate ofthe inhibitor PKF52-524 to CD45 phosphatase was estimated to be 59 M-1 s-1.