ABSTRACT
I. Introduction ...................................................................................................................... 744
A. Enzyme Catalysis ..................................................................................................... 744
B. The Chemical Step: Contributions of Quantum Mechanical Tunneling,
Equilibrium Fluctuations, and Dynamics ................................................................ 744
II. Kinetic Isotope Effects as Probes of the Chemical Step................................................. 745
A. Semiclassical Relationship of Reaction Rates of H, D, and T ............................... 746
B. Primary (18) Swain-Schaad Relationship............................................................... 746
1. Intrinsic 18 KIE ................................................................................................. 746
2. Experimental Examples Using Intrinsic 18 KIE............................................... 748
a. Peptidylglycine a-Hydroxylating Monooxygenase .................................... 748
b. Thymidylate Synthase ................................................................................. 748
c. Dihydrofolate Reductase ............................................................................. 748
C. Secondary (28) Swain-Schaad Relationship........................................................... 748
1. Mixed Labeling Experiments as Probes for Tunneling
and 18-28 Coupled Motion ............................................................................... 749
2. Upper Semiclassical Limit for 28 Swain-Schaad Relationship....................... 750
a. Zero-Point Energy and Reduced Mass Considerations .............................. 750
b. Vibrational Analysis.................................................................................... 751
c. Effect of Kinetic Complexity ...................................................................... 751
d. The New Effective Upper Limit ................................................................. 752
3. Experimental Examples Using 28 Swain-Schaad Exponents.......................... 753
a. Horse Liver Alcohol Dehydrogenase.......................................................... 753
b. Thermophilic ADH from Bacillus stearothermophilus (ADH-hT)............ 753
III. Temperature Dependence of KIEs .................................................................................. 753
A. Temperature Dependence of Reaction Rates and KIEs.......................................... 753
B. KIEs on Arrhenius Activation Factors .................................................................... 754
C. Experimental Examples Using Isotope Effects on Arrhenius
Activation Factors .................................................................................................... 755
1. Soybean Lipoxygenase-1 .................................................................................. 755
2. Thermophilic ADH (ADH-hT) ......................................................................... 756
IV. Theoretical Approaches ................................................................................................... 757
A. Phenomenological “Marcus-Like” Models ............................................................. 757
B. QM/MM Models and Simulations........................................................................... 758
V. Comparison to Studies of Nonenzymatic Reactions ....................................................... 759
VI. Conclusions ...................................................................................................................... 760
References..................................................................................................................................... 760
In biological systems, enzymes are the catalysts that direct, control and enhance chemical
transformations. Enzymes evolved to accomplish two almost contradictory tasks: one is catalyzing
a reaction at a rate most suitable for organism function and the other is preventing alternative side-
processes that would commonly occur in nonenzymatic reactions. In other words, an enzyme not
only catalyzes the reaction of interest, it also inhibits side reactions and the formation of by-
products. Commonly, the first effect is denoted as catalysis and the second is denoted as specificity.
The rate enhancement is often many orders of magnitude greater than the reaction in solution, and
of substantial biological importance.
