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• W2018441310 startingPage "559" @default.
• W2018441310 abstract "Phosphorylation is central to signal transduction in living organisms. The specificity of phosphorylation ensures signaling fidelity. Understanding substrate specificity is essential for novel assay development in drug discovery. In this study, we have developed an innovative approach to study protein kinase and its substrate specificity. Using 24 micro parallel liquid chromatography, we studied the reaction kinetics for two different peptide substrates commonly associated with protein kinase A (PKA): Kemptide (Leu-Arg-Arg-Ala-Ser-Leu-Glu) and CREBtide (Lys-Arg-Arg-Glu-Ile-Leu-Ser-Arg-Arg-Pro-Ser-Tyr-Arg). The phosphorylation of each substrate was monitored in real time, and the kinetic parameters (V(max), K(m), k(cat), and k(cat) K(m)) were determined for a variety of initial conditions. The results from several kinetic experiments indicated that Kemptide had higher V(max) and k(cat) values compared to CREBtide under the same assay conditions. However, both substrates had a similar k cat)/K(m) value, suggesting that both substrates have similar specificity constants for PKA. We further analyzed the reaction kinetics of ATP for both PKA/substrate complexes. Interestingly, we found that there was a fivefold difference in the specificity constants for ATP affinity to the two complexes, suggesting that even though the sequence differences between the two substrates do not affect their independent interactions with PKA, the differences do have a secondary effect on each enzyme's interaction with ATP and significantly alter the ATP consumption and thus phosphorylation. This novel approach has a broad application for studying enzyme functions and enzyme/substrate specificity." @default.
• W2018441310 created "2016-06-24" @default.
• W2018441310 creator A5033716777 @default.
• W2018441310 creator A5056371645 @default.
• W2018441310 creator A5074513522 @default.
• W2018441310 date "2007-08-01" @default.
• W2018441310 modified "2023-09-23" @default.
• W2018441310 title "A MicroPLC-Based Approach for Determining Kinase-Substrate Specificity" @default.
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• W2018441310 doi "https://doi.org/10.1089/adt.2007.072" @default.
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