The Enzyme That Puts the Pedal to the Metal: Metal Substituted Carbonic Anhydrase

The Enzyme That Puts the Pedal to the Metal: Metal Substituted Carbonic Anhydrase

Carbonic anhydrases (CAs; EC are a family of metalloenzymes that catalyze the reversible hydration of carbon dioxide and bicarbonate. Since their discovery in the 1930’s (Meldrum & Roughton, 1933; Stadie & O'Brien, 1933), CAs have been at the forefront of scientific discovery as these ubiquitous enzymes equilibrate the reaction between four simple molecules: water, carbon dioxide, bicarbonate, and protons. As such, these enzymes have important roles in ion transport, acid–base regulation, gas exchange, photosynthesis, CO2 fixation, and supplying CO2into many physiological processes. Hence, their studies have contributed in research from the basic understanding of enzymatic reactions, structural biology, molecular dynamics, drug discovery, and clinical medicine.

Fig. 1. Overall structure of zinc-CA II. Shown CO2located in the active site (red box).

The study presented, was born from the combined efforts of two research groups from Ulsan National Institute of Science and Technology (UNIST) S. Korea and the University of Florida (UF) USA. The initial seeds of this collaboration go back over a decade, when the Principal Investigators of the two labs first meet at Cornell University synchrotron, while working on the capture of carbon dioxide bound in the active site of human carbonic anhydrase II (CAII) (Fig. 1). A feat, that many at the time thought impossible, due the insoluble nature of carbon dioxide and the near rate-of-diffusion catalytic rate (Domsic et al, 2008).

The premise of this study was forthcoming from literature, namely that zinc–CA is the dominant circulating form, yet there is an overabundance of CA isoforms expressed in humans, with CA I and II especially abundant red blood cells, and several reports have shown CA II is a promiscuous enzyme, when it comes to its active site catalytic metal.

Hence, in this study, we wanted to examine structurally the role of metal ions in CA II, and as such we selected four divalent transition-metal ions; zinc, cobalt, nickel, and copper, that alter CA II activity from 100, ~50, ~2, and 0%, respectively (Lindskog and Nyman, 1964). To achieve this, we utilized X-ray crystallography to examine the catalytic intermediate states of the metal-free and the four metal-bound CA II by cryocooling the crystals under CO2 pressures ranging from 0 (no CO2pressurization) to 20 atm.

Fig. 2. Substrate/product binding at the active site of CA II with different metal ion substitutions.

What we found was the characteristic metal ion coordination geometries directly modulated the catalytic processes, including substrate binding, its conversion to product, and product binding (Fig. 2). In addition, surprisingly we saw that the metal ions have a long-range (~10 Å) electrostatic effect on restructuring the water network within the enzyme active site, affecting the product displacement and the proton transfer process.

We feel our experimental results can be used as direct input for theoretical and computational studies on the role of metal ions, which we anticipate could open a new window to the study of metal–protein relationships, drug discovery targeting metalloenzymes, engineering of natural metalloenzymes, rational design of de novo metalloenzymes, and synthesis of supramolecular analogues to metalloenzymes.


Meldrum, N. U. & Roughton, F. J. W.  Carbonic anhydrase. Its preparation and properties. J. Physiol. 80, 113–142, doi: 10.1113/jphysiol.1933.sp003077(1933).

Stadie, W. C. & O'Brien, H. The catalysis of the hydration of carbon dioxide and the dehydration of carbonic acid by an enzyme isolated from red blood cells. J. Biol. Chem., 103, 521–529, (1933).

Domsic, J. al.Entrapment of carbon dioxide in the active site of carbonic anhydrase II. J Biol Chem283, 30766-30771, doi:10.1074/jbc.M805353200 (2008).

Lindskog, S. & Nyman, P. O. Metal-Binding Properties of Human Erythrocyte Carbonic Anhydrases. Biochim Biophys Acta85, 462-474, doi:10.1016/0926-6569(64)90310-4 (1964).

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