TY - CHAP
T1 - Binding Isotope Effects for Interrogating Enzyme–Substrate Interactions
AU - Stratton, Christopher F.
AU - Poulin, Myles B.
AU - Schramm, Vern L.
N1 - Funding Information:
We thank Dr. D. Randal Kipp (Relay Therapeutics) for helpful discussions regarding the use of the RED device for equilibrium BIE measurements. This work was supported by research Grants GM041916 and CA135405 from the National Institutes of Health.
Publisher Copyright:
© 2017 Elsevier Inc.
PY - 2017
Y1 - 2017
N2 - Equilibrium binding isotope effects (BIEs) report on the bond vibrational status of enzyme substrates in the Michaelis complex prior to the transition state and how they differ from the solution state. Accordingly, BIEs provide an experimental means of interrogating enzyme–substrate interactions and inform on the influence of enzyme-mediated atomic distortions in modulating substrate reactivity. In this chapter, we outline a rapid equilibrium dialysis method that our lab has used to measure BIEs for several enzyme systems. Implementation of the rapid equilibrium dialysis approach is described in the context of our recent studies on the substrate bonding environment for the human protein lysine N-methyltransferase NSD2. A summary of BIE effects provides context for the range of experimental values.
AB - Equilibrium binding isotope effects (BIEs) report on the bond vibrational status of enzyme substrates in the Michaelis complex prior to the transition state and how they differ from the solution state. Accordingly, BIEs provide an experimental means of interrogating enzyme–substrate interactions and inform on the influence of enzyme-mediated atomic distortions in modulating substrate reactivity. In this chapter, we outline a rapid equilibrium dialysis method that our lab has used to measure BIEs for several enzyme systems. Implementation of the rapid equilibrium dialysis approach is described in the context of our recent studies on the substrate bonding environment for the human protein lysine N-methyltransferase NSD2. A summary of BIE effects provides context for the range of experimental values.
KW - Analysis of binding isotope effects
KW - Catalytic site distortion
KW - Equilibrium dialysis
KW - Ground-state stabilization
KW - Inhibitor binding effects
KW - Molecular distortion
KW - Transition-state analysis
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U2 - 10.1016/bs.mie.2017.07.019
DO - 10.1016/bs.mie.2017.07.019
M3 - Chapter
C2 - 28911767
AN - SCOPUS:85028586603
T3 - Methods in Enzymology
SP - 1
EP - 21
BT - Methods in Enzymology
PB - Academic Press Inc.
ER -