ABSTRACT
I. Isotope Effect Theory....................................................................................................... 915
A. Notation .................................................................................................................... 916
B. Measurement of Isotope Effects .............................................................................. 916
1. Direct Comparison ............................................................................................ 916
2. Internal Competition ......................................................................................... 917
a. The Remote Label Method — Stable Isotopes .......................................... 918
b. The Remote Label Method — Radioactive Isotopes ................................. 920
3. Equilibrium Perturbation................................................................................... 920
C. Equation for the Isotope Effect................................................................................ 921
D. Determination of Intrinsic Isotope Effects .............................................................. 923
1. Northrop’s Method ............................................................................................ 923
2. Multiple Isotope Effect Method ........................................................................ 924
II. Examples of Mechanistic Analysis.................................................................................. 925
A. Aspartate Transcarbamoylase .................................................................................. 925
B. Malic Enzyme .......................................................................................................... 926
C. Oxalate Decarboxylase ............................................................................................ 927
D. Chorismate Mutase .................................................................................................. 927
E. L-Ribulose-5-P 4-Epimerase.................................................................................... 928
F. Other Enzymes ......................................................................................................... 928
References..................................................................................................................................... 928
Isotope effects are a powerful tool for the study of enzyme mechanisms, since they look at the
chemistry while it is taking place and can give information on transition state structure. They are
caused by substituting a heavier isotope for a lighter one in a substrate for the enzyme. Isotopic
substitution can affect either the rate of a reaction (a kinetic isotope effect) or the equilibrium
constant (an equilibrium isotope effect; see Chapter 2). They are called primary isotope effects
when a bond is made or broken to the isotopic atom during the reaction, and secondary when the
bonding to this atom changes, but no bonds to it are made or broken (see Chapter 39). Secondary
isotope effects are called a if bonds are made or broken to an atom attached to the isotopic one, or b
if they are made or broken to the second atom from the isotopic one. Thus in the conversion of
ethanol to acetaldehyde, deuterium substitution in the CH
hydrogen atom transferred to NAD
during the reaction gives a primary isotope effect, while substitution in the non-transferred CH
hydrogen gives an a secondary isotope effect. Substitution in the methyl hydrogens gives a b
secondary isotope effect, and running the reaction in D
O so the OH becomes deuterated will give a
primary isotope effect.
