Ligand-induced conformational changes in ribonuclease.

The rate of formation of ribonuclease-S from ribonuclease A by the action of subtilisin was measured at pH 5.5 in the presence and absence of the strongly bound ribonuclease inhibitor, 2’-cytidylate. Presence of the inhibitor in the active center significantly reduced the rate of formation of ribonuclease-S. Since bond 20-21 (and, to a lesser extent, 21-22), the cleavage of which is responsible for the formation of this derivative, is well removed from the active center region, and thus from the binding site of the inhibitor, steric hindrance by the latter can be excluded as the cause for this reduced rate. Instead, it is concluded that the presence of the inhibitor in the active center caused a conformational change which affected a distant region of the molecule. Amino-terminal analysis confirmed cleavage at the expected major site, bond 20-21. The presence of the competitive inhibitors, 2’and 3’cytidylate, afforded considerable protection against inactivation of ribonuclease A by trypsin and by chymotrypsin, when digested at 60”. End group analysis confirmed that the bonds split were those expected from previous work of others. The presence of 2’-cytidylate completely prevented the temperature-induced optical rotatory change characteristic of ribonuclease, indicating a high degree of structural stabilization by the ligand. The data presented support the view that the presence of the competitive inhibitor in the active center region alters the proteolytic susceptibility of bonds at multiple sites, situated outside of this region.