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.