Fluorescent inhibitor probes of enzyme active site conformation: anion binding to angiotensin-converting enzyme.

Dansylated tight-binding inhibitors are effective fluorophoric probes for detecting conformational changes of enzyme active sites. In this study they have been employed to examine the effect of anions on the conformation of angiotensin-converting enzyme. The efficiency of radiationless energy transfer between enzyme tryptophan residues and an active site-bound dansyl inhibitor has been shown to be enhanced by the addition of chloride. Half-maximal fluorescence enhancement occurs at about 2 mM chloride and is the same for both N-(1-carboxyl-5-dansylamino-pentyl)-glycyl-L-phenylalanine [Ki,app = 50 nM (pH 7.5, 300 mM NaCl)] and N-(1-carboxyl-5-dansylamino-pentyl)-glycyl-L-lysine (Ki,app = 5.7 nM). Other activating anions also evoke similar increases in enzyme-inhibitor energy transfer. Fluorescence changes are not due to binding additional inhibitor molecules but rather to an anion-induced change in protein conformation.