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• W4377082013 abstract "We describe the experimental methods and analysis to define the role of enzyme conformational changes in specificity based on published studies using DNA polymerases as an ideal model system. Rather than give details of how to perform transient-state and single-turnover kinetic experiments, we focus on the rationale of the experimental design and interpretation. We show how initial experiments to measure kcat and kcat/Km can accurately quantify specificity but do not define its underlying mechanistic basis. We describe methods to fluorescently label enzymes to monitor conformational changes and to correlate fluorescence signals with rapid-chemical-quench flow assays to define the steps in the pathway. Measurements of the rate of product release and of the kinetics of the reverse reaction complete the kinetic and thermodynamic description of the full reaction pathway. This analysis showed that the substrate-induced change in enzyme structure from an open to a closed state was much faster than rate-limiting chemical bond formation. However, because the reverse of the conformational change was much slower than chemistry, specificity is governed solely by the product of the binding constant for the initial weak substrate binding and the rate constant for the conformational change (kcat/Km = K1k2) so that the specificity constant does not include kcat. The enzyme conformational change leads to a closed complex in which the substrate is bound tightly and is committed to the forward reaction. In contrast, an incorrect substrate is bound weakly, and the rate of chemistry is slow, so the mismatch is released from the enzyme rapidly. Thus, the substrate-induced-fit is the major determinant of specificity. The methods outlined here should be applicable to other enzyme systems." @default.
• W4377082013 created "2023-05-20" @default.
• W4377082013 creator A5054934658 @default.
• W4377082013 creator A5079509051 @default.
• W4377082013 date "2023-01-01" @default.
• W4377082013 modified "2023-10-18" @default.
• W4377082013 title "Design and interpretation of experiments to establish enzyme pathway and define the role of conformational changes in enzyme specificity" @default.
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• W4377082013 doi "https://doi.org/10.1016/bs.mie.2023.03.018" @default.
• W4377082013 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/37245912" @default.
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