ABSTRACT
I. Introduction ...................................................................................................................... 794
A. Nomenclature ........................................................................................................... 794
B. Types of Isotope Effects .......................................................................................... 794
II. Enzyme-Catalyzed vs. Nonenzymatic Reactions ............................................................ 794
A. Physical vs. Chemical Steps .................................................................................... 794
B. Commitment Factors................................................................................................ 795
C. Substrate Stickiness.................................................................................................. 795
III. Substrate Dependence of Isotope Effects ........................................................................ 796
A. Sequential Mechanisms............................................................................................ 796
1. Ordered Mechanisms (k
, k
, k
, and k
¼ 0)................................................... 797
a. Formate Dehydrogenase.............................................................................. 797
b. Mannitol Dehydrogenase ............................................................................ 798
2. Random Mechanisms (All Rate Constants of
Mechanism 3 Apply)......................................................................................... 798
a. NAD-Malic Enzyme.................................................................................... 799
b. Ketopantoate Reductase .............................................................................. 799
B. Ping Pong Kinetic Mechanisms............................................................................... 800
1. Dihydroorotate Dehydrogenase......................................................................... 801
2. p-Cresol Methylhydroxylase ............................................................................. 801
C. Substrate Dependence of Isotope Effects in Terreactant
and Higher Order Mechanisms................................................................................ 801
1. NAD-Malic Enzyme.......................................................................................... 802
2. Alanine Dehydrogenase .................................................................................... 802
IV. pH Dependence of Isotope Effects .................................................................................. 802
A. Proton Transfer and Chemistry are Concerted........................................................ 802
1. Random Addition of Proton and Substrate to Enzyme .................................... 803
a. NADP-Malic Enzyme ................................................................................. 804
b. Nitroalkane Oxidase .................................................................................... 804
2. Dead-End Protonation of Enzyme .................................................................... 804
a. NAD-Malic Enzyme.................................................................................... 805
3. Dead-End Protonation of Enzyme and the Enzyme-Reactant Complex ......... 805
a. Ketpantoate Reductase ................................................................................ 805
4. Dead-End Formation of a Protonated Enzyme-Reactant Complex ................. 806
B. Proton Transfer and Chemistry not Concerted........................................................ 806
1. Equine Liver Alcohol Dehydrogenase.............................................................. 807
V. Closing Remarks .............................................................................................................. 807
References..................................................................................................................................... 808
Isotope effects are perhaps one of the most powerful tools available to the mechanistic investigator
based on the amount and different types of information one can obtain. In this short treatise, an
overview of isotope effects applied to the determination of the kinetic mechanism of enzymatic
reactions via the substrate and pH dependence of isotope effects will be provided.
