A Deuterium-Hydrogen Exchange Study of Inhibitor-Induced Conformational Changes in Ribonuclease A.

Deuterium-hydrogen exchange has been used to detect possible conformational changes accompanying the interaction of ribonuclease A with two competitive inhibitors cytidine 3'- phosphate (Cyd-3'-P) and cytidine 2'-phosphate (Cyd-2'-P). An improvement of the cryosublimation technique was employed and combined. with mass spectrometry measurements of sample deuterium content. Exchange kinetics were followed from 1 min to 24 h, at pH 6.5, and 38&degree;C (0.1 M NaCl). Two methods were used to present the data: a graphical exponential-curve-fitting and a numerical analysis. They both lead to the same conclusions. Four different classes of hydrogens were found. Among them, the one involving the instantaneously exchanged atoms is increased by about ten hydrogens after the binding of these inhibitors, the number of unexchanged hydrogens (within 24 b) is hardly altered in the presence of Cyd-3'-P whereas it is decreased by one third after the fixation of Cyd-2'-F. These exchange results suggest that both inhibitors induce in ribonuclease A conformational changes which may be correlated with inhibitor strength. [ABSTRACT FROM AUTHOR]