Backbone dynamics of Fusarium solani pisi cutinase probed by nuclear magnetic resonance: The lack of interfacial activation revisited

The backbone dynamics of Fusarium solani pisi cutinase has been studied by a variety of nuclear magnetic resonance experiments to probe internal motio ns on different time scales. The core of cutinase appears to be highly rigi d. The binding site, including the oxyanion hole, is mobile on the microsec ond to millisecond time scale, in contrast to the well-defined active site and preformed oxyanion hole elucidated by X-ray crystallography [Martinez, C., de Geus, P., Lauwereys, M., Matthyssens, G., and Cambillau, C. (1992) N ature 356, 615-618]. In this crystal structure, cutinase has a rather open conformation, in which the hydrophobic binding site is exposed. The observe d mobility in solution most likely represents the interconversion between o pen and more closed conformations, like in a true lipase. The opening and c losing motions are on a time scale which corresponds with the kinetics of t he hydrolysis reaction, i.e,, the millisecond range, which suggests that th ese conformational rearrangements form the rate-limiting step in catalysis, We conclude that the crystal structure probably represents one of the mult iple conformations present in solution, which fortuitously is the active co nformation. The implications of our findings are discussed with particular reference to the explanation of the lack of interfacial activation as found for cutinase.