Authors: Stepan B. Lesnichin†‡, Ilya G. Shenderovich*†‡, Titin Muljati†, David Silverman, and Hans-Heinrich Limbach*†
Journal: Journal of the American Chemical Society
Affiliation: †Institut für Chemie und Biochemie, Freie Universität Berlin
‡Department of Physics, St. Petersburg State University
Departments of Pharmacology and Therapeutics, and Biochemistry and Molecular Biology, College of Medicine, University of Florida
Carbonic anhydrase has a very important role. It catalyzes the reversible hydration of CO2, important for regulating pH balance in animals. The enzyme uses a zinc ion to tune the acidity of a zinc-bound water molecule, enabling the combination of CO2 with OH– to form and release bicarbonate (HCO3–) (see below). The local histidine is 7 Å away from the zinc ion, suggesting that protons are transferred from the water bound zinc to the histidine by a network of hydrogen-bonded water molecules connecting the two sites.
where E = enzyme and B = a local histidine residue that acts as a base.
A variety of synthetic models have attempted to mimic the acid-base chemistry of zinc-bound water molecules in aqueous solution. However, the local environment of the protein active site has only a few water molecules, and so these small-molecule mimics may not accurately model the acidities of the zinc-bound water. Thus, in this report, the authors examined water-bound trispyrazolylhydroborate zinc complexes in dichloromethane (a polar, aprotic solvent) as potential models.
The authors focused on three complexes (3, 4, and 5) depicted in the figure at the top. In 3, the zinc-bound hydroxo group forms a hydrogen bond with 2,4,6-trimethylpyridine (collidine or Col). They chose Col because its protonated form has a pKa value very similar to protonated 4-methylimidazole (and hence similarity to histidine residues in proteins). For complex 4, they used [(C6F5)3BOH2] instead of Col as a comparison. Finally, the complex between Col and [(C6F5)3BOH2] (5) was examined to check the relative proton donating ability of the two species.
Using 1H-NMR and 15N-NMR (with the 15N derivatives), the authors first confirmed the existence of the hydrogen bonds and then investigated the properties of these hydrogen bonds (details discussed in the paper). Comparing with acidities of carboxylic acids, they were able to infer a pKa of 2.2 (in water) for the zinc-bound water in their complex.
Other studies that have found that hydrogen bonds between acids and bases in enzyme active sites act similarly in polar aprotic solution. Thus, this study concludes that, although their model is very different from carbonic anhydrase, using a polar aprotic solvent may provide better models for the proton exchange in carbonic anhydrase.