ABSTRACT
I. Introduction ...................................................................................................................... 847
A. Catalytic Mechanism................................................................................................ 848
B. Structure ................................................................................................................... 849
C. Relevance of Ordered Water in Crystal Structures
of Carbonic Anhydrase ............................................................................................ 850
II. Isotope Effects on First Stage of Catalysis — The Hydration of CO
........................... 850
III. Solvent Hydrogen Isotope Effects on the Proton Transfer Steps ................................... 850
A. Intramolecular Proton Transfer in Catalysis by Carbonic Anhydrase.................... 850
1. Proton inventory ................................................................................................ 850
2. Interpreting the Isotope Effects......................................................................... 851
3. Theorists View of Isotope Effects in Catalysis by Carbonic Anhydrase......... 851
4. Use of
O Exchange ........................................................................................ 852
5. Marcus Rate Theory Allows Enhanced Interpretation
of Proton-Transfer Rates ................................................................................... 853
6. Marcus Plot for Intramolecular Proton Transfer .............................................. 854
7. Marcus Plot for Solvent Hydrogen Isotope Effects.......................................... 855
B. Intermolecular Proton Transfer in Catalysis by Carbonic Anhydrase.................... 856
1. Marcus Plot for Solvent Hydrogen Isotope Effects.......................................... 856
2. The Marcus Formalism Extended to the b and g Classes ............................... 857
VI. Conclusions ...................................................................................................................... 857
References..................................................................................................................................... 857
The seminal study of isotope effects in the catalysis of CO
hydration by carbonic anhydrase laid
the framework not only for many future studies of isotope effects in this pathway but also for an
isotopic approach to understanding proton transfer through water bridges. This was the 1975 report
of Steiner, Jonsson, and Lindskog
demonstrating the solvent hydrogen isotope effect of 3.8 on the
maximal velocity of catalysis by carbonic anhydrase, an isotope effect that was correctly interpreted
as arising from an intramolecular proton transfer. The current review expands on this and related
investigations and is centered on understanding proton transfers through intervening water
molecules in a protein environment. Detailed crystal structures are now available for very complex
systems in which proton transport through distances plays a significant role, such as
bacteriorhodopsin, cytochrome c oxidase, and the bacterial reaction center. In these cases the
proton-transfer pathway may extend up to 15 or 20 A
˚
and involve intervening water and amino-acid
residues, a tortuous pathway in which often the identity of the proton acceptor and donor may be in
doubt. Catalysis by carbonic anhydrase is a very useful model to study such processes since the
identity of the proton donor and acceptor is clear, the rate constant for proton transfer limits
maximal velocity, and the pathway involves intervening water. The crystal structures of carbonic
anhydrase show ordered water between the proton donor and acceptor, often in hydrogen-bonded
chains although the relevance of this to proton transfer is not known. Moreover, there is a very
broad range of isozymes of the carbonic anhydrases (CAs) p to work with including three classes
of structurally unrelated carbonic anhydrases, examples of convergent evolution. The enzymes in
these classes are all zinc-metalloenzymes but bear no amino-acid homologies between classes
: the
a class which includes the animal and human CAs, the b class which includes the plant and many
bacterial CAs, and the g class which includes an archaeal CA. The catalytic mechanism of the
carbonic anhydrases has been the subject of many reviews.
