Competitive Inhibitory Effects of Acetazolamide upon Interactions with Bovine Carbonic Anhydrase II |
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Authors: | Shahrokh Safarian Fatemeh Bagheri Ali Akbar Moosavi-Movahedi Massoud Amanlou Nader Sheibani |
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Institution: | (1) Department of Cell and Molecular Biology, School of Biology, University College of Science, University of Tehran, Tehran, Iran;(2) Institute of Biochemistry & Biophysics, University of Tehran, Tehran, Iran;(3) Faculty of Pharmacology, Tehran University of Medical Sciences, Tehran, Iran;(4) Departments of Ophthalmology and Visual Sciences and Pharmacology, University of Wisconsin, Madison, WI, USA |
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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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Keywords: | Bovine carbonic anhydrase II acetazolamide binding study competitive inhibition isoforms |
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