ABSTRACT
I. Introduction ...................................................................................................................... 995
II. Origins of Solvent Isotope Effects................................................................................... 996
A. Equilibrium Solvent Isotope Effects........................................................................ 996
B. Kinetic Isotope Effects............................................................................................. 997
III. The Kresge-Gross-Butler Equation............................................................................... 998
A. Derivation................................................................................................................. 998
B. The Proton Inventory Technique........................................................................... 1000
1. Proton Inventories of Elementary Steps ......................................................... 1000
2. Effects of Reactant State Fractionation .......................................................... 1001
3. Proton Inventories of Multistep Enzyme Reactions....................................... 1002
IV. Fractionation Factors...................................................................................................... 1004
A. Reactant State Fractionation Factors of Common Functional Groups ................. 1004
B. Transition State Fractionation Factors................................................................... 1005
V. Practical Considerations................................................................................................. 1006
VI. Examples ........................................................................................................................ 1008
A. Nonenzymic Reactions .......................................................................................... 1008
B. Enzymatic Reactions.............................................................................................. 1009
1. Serine Proteases............................................................................................... 1009
2. Acetylcholinesterase........................................................................................ 1012
3. Carbonic Anhydrase ........................................................................................ 1014
4. Tyrosine Hydroxylase ..................................................................................... 1015
VII. Conclusions .................................................................................................................... 1016
References................................................................................................................................... 1016
Solvent isotope effects arise in enzymatic reactions when rate or equilibrium constants are
measured in H
O, D
O, or mixtures of these isotopic solvents. Since there are multiple isotopically
exchangeable sites in an enzyme reaction assembly (i.e., enzyme þ substrate), the origins of
solvent isotope effects are conceivably of vexing complexity. The multisite origin of solvent
isotope effects is suggested by the wide range of isotopic fractionation factors observed in
staphylococcal nuclease
(fractionation factors are discussed later in this chapter). However, like all
isotope effects, when solvent isotope effects and their interpretation are corroborated by additional
experimental results, they are of considerable utility in elucidating the elements of structure and
function that contribute to enzyme catalytic power. This chapter outlines the conceptual basis of
solvent isotope effects, discusses practical considerations when measuring and interpreting them,
and explores the use of solvent isotope effects in several enzyme-catalyzed reactions. The
application of solvent isotope effects to enzyme reactions has been extensively reviewed.
