Molecular Pharmacology, Vol 12, 832-843, Copyright © 1976 by the American Society for Pharmacology and Experimental Therapeutics

A Comparative Study of Mammalian Erythrocyte Carbonic Anhydrases Employing Spin-Labeled Analogues of Inhibitory Sulfonamides

¹ Section on Molecular Pharmacology, Pulmonary Branch, National Heart and Lung Institute, Bethesda, Maryland 20014
² Division of Computer Research and Technology, National Institutes of Health, Bethesda, Maryland 20014
³ Department of Human Genetics, University of Michigan Medical School, Ann Arbor, Michigan 48104

The topography of the active sites of rhesus carbonic anhydrases I and II, rabbit carbonic anhydrase I, sheep carbonic anhydrase II, and dog carbonic anhydrase I has been studied with the aid of spin-labeled analogues of acetazolamide and sulfanilamide. Electron spin resonance measurements indicated that the pyrrolidine ring of 2,2,5,5-tetramethyl-3-[(p-sulfamoylphenyl)carbamoyl]-1-pyrrolidinyloxyl became highly immobilized when this label bound to the active site of rhesus carbonic anhydrase I. As the chain length between the aromatic and pyrrolidine rings was increased, the mobility of the nitroxide group of the enzyme-bound inhibitor progressively increased, until with 2,2,5,5-tetramethyl-3-({[(p-sulfamoylphenyl)carbamoyl]methyl}carbamoyl)-1-pyrrolidinyloxyl there was only minimal interaction between the heterocyclic ring and the active site of rhesus carbonic anhydrase I. These findings suggest that the active site of rhesus carbonic anhydrase I is a cleft about 14 A deep. Similar experiments indicated that the topography of the active sites of rhesus carbonic anhydrase II, dog carbonic anhydrase I, and sheep carbonic anhydrase II were similar to that of rhesus carbonic anhydrase I, while the active site of rabbit carbonic anhydrase was somewhat deeper. Spin-labeled inhibitor 2,2,6,6-tetramethyl-4-(p-sulfamoylbenzamide)piperidinooxyl became highly immobilized on binding to type II (high-activity) carbonic anhydrase but exhibited isotropic motion at the active sites of type I (low-activity) isozymes. An attempt is made to explain these results in terms of the three-dimensional structure of the active sites of human carbonic anhydrases I and II.

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ACKNOWLEDGMENTS The authors are greatly indebted to Drs. K. K. Kannan, I. Waara, and co-workers of the University of Uppsala, Sweden, for making available the crystal coordinates for human carbonic anhydrases I and II before publication.