chapter  31
26 Pages

Catalysis by Alcohol Dehydrogenases

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.