Abstract
Sulfonamide drugs mediate their main therapeutic effects through modulation of the activity of membrane and cytosolic carbonic anhydrases. How interactions of sulfonamide drugs impact structural properties and activity of carbonic anhydrases requires further study. Here the effect of acetazolamide on the structure and function of bovine carbonic anhydrase II (cytosolic form of the enzyme) was evaluated. The Far-UV CD studies indicated that carbonic anhydrase, for the most part, retains its secondary structure in the presence of acetazolamide. Fluorescence measurements using iodide ions and ANS, along with ASA calculations, revealed that in the presence of acetazolamide minimal conformational changes occurred in the carbonic anhydrase structure. These structural changes, which may involve spatial reorientation of Trp 4 and Trp 190 or some other related aminoacyl residues near the active site, considerably reduced the catalytic activity of the enzyme while its thermal stability was slightly increased. Our binding results indicated that binding of acetazolamide to the protein could occur with a 1:1 ratio, one mole of acetazolamide per one mole of the protein. However, the obtained kinetic results supported the existence of two acetazolamide binding sites on the protein structure. The occupation of each of these binding sites by acetazolamide completely inactivates the enzyme. Advanced analysis of the kinetic results revealed that there are two substrate (p-NPA) binding sites whose simultaneous occupation is required for full enzyme activity. Thus, these studies suggest that the two isoforms of CA II should exist in the medium, each of which contains one substrate binding site (catalytic site) and one acetazolamide binding site. The acetazolamide binding site is equivalent to the catalytic site, thus, inhibiting enzyme activity by a competitive mechanism.
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Abbreviations
- CA:
-
carbonic anhydrase
- BCA:
-
bovine carbonic anhydrase
- HCA:
-
human carbonic anhydrase
- p-NPA:
-
p-nitrophenylacetate
- Trp:
-
tryptophan
- His:
-
histidine
- ε:
-
extinction coefficient
- ANS:
-
anilinonaphtalene sulfonic acid
- KI:
-
potassium iodide
- CD:
-
circular dichroism
- ASA:
-
accessible surface area
- mAb/min:
-
milliabsorbance per minute
- a.u.:
-
arbitrary unit
- Å2 :
-
surface unit equal to one square of Angstrom
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Acknowledgments
Financial support for this work was provided by the Research Council of the University of Tehran. The authors would like to thank Dr. Christine Sorenson (University of Wisconsin, Madison, Wisconsin) for valuable comments. The authors would like to thank the Sealy Center for Structural Biology at the University of Texas Medical Branch and the RCSB organization for their valuable web service.
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Safarian, S., Bagheri, F., Moosavi-Movahedi, A.A. et al. Competitive Inhibitory Effects of Acetazolamide upon Interactions with Bovine Carbonic Anhydrase II. Protein J 26, 371–385 (2007). https://doi.org/10.1007/s10930-007-9073-4
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DOI: https://doi.org/10.1007/s10930-007-9073-4