NMR studies of Fusarium solani pisi cutinase in complex with phosphonate inhibitors

The backbone dynamics of Fusarium solani pisi cutinase in complex with a ph osphonate inhibitor has been studied by a variety of nuclear magnetic reson ance experiments to probe internal motions on different time scales. The re sults have been compared with dynamical studies performed on free cutinase. In solution, the enzyme adopts its active conformation only upon binding t he inhibitor. While the active site Ser(120) is rigidly attached to the sta ble alpha/beta core of the protein, the remainder of the binding site is ve ry flexible in the free enzyme. The other two active site residues Asp(175) and His(188) as well as the oxyanion hole residues Ser(42) and Gln(121) ar e only restrained into their Proper positions upon binding of the substrate -like inhibitor. The flap helix, which opens and closes the binding site in the free molecule, is also fixed in the cutinase-inhibitor complex. Our re sults are in contrast with the X-ray analysis results, namely that in the p rotein crystal, free cutinase has a well-defined active site and a preforme d oxyanion hole and that it does not need any rearrangements to bind its su bstrate. Our solution studies show that cutinase does need conformational r earrangements to bind its substrate, which may form the rate-limiting step in catalysis.