NMR Spectroscopic Investigation of Human Carbonic Anhydrase Points to Alternative Mechanism of Action
Carbonic anhydrase (CA) catalyzes a deceptively simple chemical reaction-the hydration of CO2 to yield bicarbonate and a proton:
CO2 + H2O = HCO3- + H+.
CA was the first enzyme recognized to have a biological function for Zn2+, which was postulated as the activation of bound H2O. Abundant in all mammalian tissues, plants, algae, and bacteria, CA is perhaps the most studied metalloprotein with more than 180 crystal structures in the protein database. Scientists at Pacific Northwest National Laboratory (PNNL) have now found evidence that a commonly held mechanism of action for human CA may not be correct. Instead, the new data are consistent with an alternative mechanism proposed by Merz, Hoffmann, and Dewar1. The evidence was generated using novel low-temperature solid-state nuclear magnetic resonance (NMR) spectroscopy, which allowed direct observation of the Zn2+ in CA.
The most active form of CA has very fast reaction rates approaching ~106/s. As a result, there have been innumerable studies directed at this protein's action mechanism. The accepted reaction mechanism holds that the water ionizes (rate-limiting step) to yield a bound hydroxide that then adds to the nearby CO2 resulting in Zn2+-bound bicarbonate. The presence of bound water and fast catalytic rates together presents a problem that up to now could not be explained. Simple arguments would predict the fastest that CA could turn over would be at rates on the order of 104/s.
The principal observable in a solid-state 67Zn NMR experiment is the quadrupole coupling constant, Cq. The 67Zn Cq values are sensitive to changes in structure and bonding associated with water or hydroxide. The accompanying figure shows data collected on the 18.8-Tesla NMR spectrometer in DOE's Environmental Molecular Sciences Laboratory illustrating that the 67Zn NMR measurement is independent of pH over the range of 5 to 8.5. In the figure, the 67Zn NMR spectrum of CA is shown at pH 5 and 8.5.
These are the low-temperature (10K) solid-state 67Zn NMR spectra of CAII at (a) pH 5 and (b) pH 8.5. Above each experimental spectrum is a simulation of the spikelet envelope. At pH 5, the extracted value of Cq is 9.6 MHz, whereas at pH 8.5 the value is 10 MHz. Full Image
In the figure, the two spectra are essentially the same. At pH 5, the Zn2+ should be coordinated by H2O, and as a result, the 67Zn NMR spectrum is expected to be three to five times broader than the spectrum at pH 8.5. This is clearly not the case; however, these data are consistent with OH- being bound to Zn2+, not H2O over the entire pH range investigated and as modeled by molecular theory. This observation is contradictory to the accepted mechanism. However, it is consistent with an alternative mechanism proposed by Merz, Hoffmann, and Dewar. Further, these data also provide an explanation for the issues associated with the turnover rates for CA. In addition, these results point to an important complementary aspect of NMR methods to X-ray diffraction, namely the sensitivity of the NMR parameters to the presence or absence of protons.
Moreover, this work demonstrates the impact zinc spectroscopy can have with respect to delineating the structure and action mechanism of this important class of metalloproteins.
These results were recently published by PNNL staff (Lipton, A. S., R. W. Heck, and P. D. Ellis. 2004. "Zinc Solid-state NMR Spectroscopy of Human Carbonic Anhydrase: Implications for the Enzymatic Mechanism." Journal of the American Chemical Society 126(14):4735-4739). This work was supported by DOE's Office of Biological and Environmental Research and by the National Institutes of Health.
1. Merz KM, R Hoffmann, and MJS Dewar. 1989. "The Mode of Action of Carbonic Anhydrase." Journal of the American Chemical Society 111(15):5636-49. DOI:10.1021/ja00197a021.