ABSTRACT
I. Introduction .................................................................................................................... 1020
II. History of Enzymatic Binding Isotope Effects.............................................................. 1020
III. Contributions of Binding and Prebinding Steps to Isotope Effects in Enzymology .... 1021
A. BIE and KIE........................................................................................................... 1022
1. Expressions of
(k
/K
) and
(k
/K
)........................................................ 1022
2. Expressions of
k
......................................................................................... 1024
B. Prebinding Isomeric Isotope Effects and KIE....................................................... 1026
1. Effect on Competitive (k
/K
) KIE Measurements...................................... 1026
a. Regimes I-III ............................................................................................ 1027
b. Regimes IV-VI......................................................................................... 1027
c. Regimes VII-IX ....................................................................................... 1028
2. Effect on Noncompetitive
k
Measurements .............................................. 1029
3. Curtin-Hammet Principle............................................................................... 1029
C. Prebinding Isotope Effects and BIE ...................................................................... 1030
D. Conclusions ............................................................................................................ 1032
1. Transition State Studies................................................................................... 1032
2. Determination of Rate-Limiting Steps and Tunneling ................................... 1032
IV. Physical Basis for Binding and Kinetic Isotope Effects ............................................... 1032
A. Frequency Changes due to Reaction and Heavy-Atom Labeling......................... 1033
1. Heavy Atom Labeling ..................................................................................... 1033
2. High-Frequency CH Bond Stretch: Equilibrium Isotope Effects................... 1034
3. Lower-Frequency CN Bond Stretch: Equilibrium Isotope Effects ................ 1035
4. When Does MMI Count?................................................................................ 1036
5. When Does EXC Count? ................................................................................ 1036
B. Alteration in Force Constants ................................................................................ 1037
1. How Many Modes Actually Matter? .............................................................. 1038
2. Isotope Effects from Altering Mode Coupling Partners................................. 1039
3. Sterics and Hyperconjugation ......................................................................... 1042
C. Summary ................................................................................................................ 1045
V. Example: Glucose and Brain Hexokinase ..................................................................... 1046
A. Methods .................................................................................................................. 1046
B. The Binary Complex.............................................................................................. 1047
C. The Ternary Complex............................................................................................ 1048
VI. Applications for BIE ...................................................................................................... 1049
VII. Conclusion...................................................................................................................... 1049
Acknowledgments ...................................................................................................................... 1050
References................................................................................................................................... 1050
Reaction rates in both chemistry and enzymology are subject to the isotopic composition of the
reactants.
