Backbone dynamics of Escherichia coli thioesterase/protease I: Evidence ofa flexible active-site environment for a serine protease

Escherichia coli thioesterase/protease I (TEP-I) is a member of a novel sub class of the lipolytic enzymes with a distinctive GDSLS motif. In addition to possessing thioesterase and protease activities, TEP-I also exhibits ary lesterase activity. We have determined the N-15 nuclear magnetic spin relax ation rates, R-1 and R-2, and the steady state H-1-N-15 heteronuclear Overh auser effect, measured at both 11.74 T and 14.09 T, of (u-N-15) TEP-I. Thes e data were analyzed using model-free formalism (with axially symmetric rot ational diffusion anisotropy) to extract the backbone dynamics of TEP-I. Th e results reveal that the core structure of the central beta -sheet and the long alpha -helices are rigid, while the binding pocket appears to be rath er flexible. The rigid core serves as a scaffold to anchor the essential lo ops, which form the binding pocket. The most flexible residues display larg e amplitude fast (ps/ns time-scale) motion and lie on one stripe whose orie ntation is presumed to be the ligand-binding orientation. We also detected the presence of several residues displaying slow (mus/ms time-scale) confor mational exchanging processes. These residues lie around the binding pocket and are oriented perpendicularly to the orientation of the flexible stripe . Two of the putative catalytic triads, Ser10 and His157, and their neighbo rs show motion on the mus/ms time-scale, suggesting that their slow motion may have a role in catalysis, in addition to their possible roles in ligand binding. The presence of a flexible substrate-binding pocket may also faci litate binding to a wide range of substrates and confer the versatile funct ional property of this protein. (C) 2001 Academic Press.