Toward the Design of Ribonuclease (RNase) Inhibitors: Ion Effects on the Thermodynamics of Binding of 2â²-CMP to RNase A
Ribonucleases (RNases) possess a variety of biological activities and, under certain conditions, are deleterious. Hence, design of selective inhibitors has been suggested as a strategy for treating RNase-related disorders. In the present study, isothermal titration calorimetry was used to measure io...
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Published in | The Journal of pharmacology and experimental therapeutics Vol. 301; no. 3; p. 925 |
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Main Authors | , , , |
Format | Journal Article |
Language | English |
Published |
American Society for Pharmacology and Experimental Therapeutics
01.06.2002
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Online Access | Get full text |
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Summary: | Ribonucleases (RNases) possess a variety of biological activities and, under certain conditions, are deleterious. Hence, design
of selective inhibitors has been suggested as a strategy for treating RNase-related disorders. In the present study, isothermal
titration calorimetry was used to measure ion effects on binding thermodynamics of the RNase A competitive inhibitor 2â²-CMP
as a representative system. The reaction cell (37°C) contained dialyzed RNase A (0.04â0.05 mM) in buffered solution (pH 5.5)
of 50 mM Na + , K + , Ca 2+ , or Mg 2+ acetate, verified spectrophotometrically. Thirty-five sequential injections (4 μl each, 3 min apart) were made of 2â²-CMP (1.2
mM) in ion-matching buffer. The data were corrected for heat of dilution. There was a 1:1 interaction in each case. The estimated
parameters (±S.D.) were: K d = 4.84 ± 0.29 μM (Na + ); 5.62 ± 0.98 μM (K + ); 24.44 ± 6.96 μM (Ca 2+ ); 28.74 ± 0.43 μM (Mg 2+ ); Î G o = â7.541 ± 0.037 kcal/mol (Na + ); â7.458 ± 1.03 kcal/mol (K + ); â6.574 ± 0.173 kcal/mol (Ca 2+ ); â6.442 ± 0.009 kcal/mol (Mg 2+ ); Î H o = â22.357 ± 1.189 kcal/mol (Na + ); â21.917 ± 0.891 kcal/mol (K + ); â20.223 ± 1.503 kcal/mol (Ca 2+ ); â26.570 ± 1.579 kcal/mol (Mg 2+ ); and Î S o = â0.048 ± 0.004 kcal/mol-K (Na + ); â0.047 ± 0.003 kcal/mol-K (K + ); â0.044 ± 0.005 kcal/mol-K (Ca 2+ ); â0.065 ± 0.005 kcal/mol-K (Mg 2+ ). Thus, all reactions were enthalpy-driven. Despite a 5-fold difference in K d between mono- and divalent ions, the ratio of ion hydration Î G o to K d was constant. These data should be useful for molecular modeling and suggest that inhibitor activity will be a function of
cellular conditions (normal or pathological). |
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ISSN: | 0022-3565 1521-0103 |
DOI: | 10.1124/jpet.301.3.925 |