ABSTRACT
I. Introduction ...................................................................................................................... 811
II. Mechanism and Structure of Alcohol Dehydrogenases .................................................. 812
A. Kinetic Mechanism from Isotope Effects ................................................................ 812
B. Structural Studies of Ternary Complexes ............................................................... 813
III. Transient Kinetics and Simulation of Complete Mechanisms........................................ 814
IV. Unmasking Chemistry for Mechanistic Studies .............................................................. 816
A. Poor Substrates and Chemical Modification ........................................................... 816
B. Site-Directed Mutagenesis ....................................................................................... 816
C. Isotope Effects and Altered Rate Constants ............................................................ 818
V. Dynamics of Hydrogen Transfer ..................................................................................... 822
A. Tunneling Detected by Comparison of H/D/T Isotope Effects .............................. 822
B. Temperature Dependence and Isotope Effects ........................................................ 822
C. Pressure and Isotope Effects .................................................................................... 825
D. Protein Motions and Computational Studies........................................................... 826
VI. Solvent (D
O) Isotope Effects and Proton Transfer........................................................ 827
A. Steady-State Kinetic Studies.................................................................................... 827
B. Transient Kinetic Studies and a Low-Barrier Hydrogen Bond............................... 827
VII. Future Studies................................................................................................................... 828
Acknowledgments ........................................................................................................................ 830
References..................................................................................................................................... 830
Determination of kinetic (substrate) and solvent isotope effects can provide important information
about which steps are rate limiting in an enzyme mechanism and about the chemistry of the
catalytic reaction. Interpretation of the isotope effects usually requires a comprehensive study of
the steady-state and transient kinetics, including pH dependences and substrate specificity, and may
be facilitated by the study of site-directed mutations that make the steps involving chemistry rate
limiting. Knowledge of three-dimensional structures of the enzyme and its complexes is useful for
interpretation of results and the design of particular mutations. In this chapter, applications of
isotope effects for study of catalysis by horse liver and yeast alcohol dehydrogenases (ADH
) will
be considered as an example of the approaches that have led to insights into the mechanisms of
proton and hydride transfer. The results suggest that a low-barrier hydrogen bond stabilizes the
ground state of the Michaelis complex for the oxidation of alcohols and that hydride is transferred
with quantum mechanical tunneling involving protein motions.
