Departamento de Cristalografía y Biología Estructural, Intituto "Rocasolano"; CSIC, Serrano 119, 28006-Madrid, Spain.

The concept of lipase interfacial activation stems from the finding that the catalytic activity of most lipases depends on the aggregation state of their substrates. It is thought that activation involves the unmasking and structuring of the enzyme's active site through conformational changes requiring the presence of oil-in-water droplets. We have reported the X-ray structure of the porcine pancreatic lipase (PL)-colipase (CL) complex obtained in the presence of the C8E4 non-ionic detergent (Hermoso et al., 1996) (Abstract), [PDF]. In that structure pancreatic lipase was found to be in the open conformation, result we attributed to the presence of detergent micelles in the crystallization medium. We have reported the neutron structure of the activated lipase/colipase/micelle complex as determined using the D2O/H2O contrast variation low resolution diffraction method (Hermoso et al. 1997) (Abstract),[PDF] and (Pignol et al., 1998) (Abstract). In the ternary complex (Fig.1), the disk-shaped micelle interacts extensively with the concave face of colipase and the distal tip of the C-terminal domain of lipase. This is , to our knowledge, the first time a micelle is visualized as a part of an enzymatic complex without displaying extensive hydrophobic interaction with the protein moieties, as in membrane proteines.

Fig. 1. The PL-CL-Micelle complex. The neutron scattering-lenght map was calculated at the protein match point (41% D2O) and contoured a 1s level. PL (gold) and CL (red) are depicted as CPK models. Reactive Serine being in green. The shape and size of the observed micelle (an ellipsoid with semi-axes of 23Åx13Åx11Å) correspond to those expected for C8E4.

Since the micelle and substrate binding sites concern different regions of the protein complex, we conclude that lipase activation is not interfacial but occurs in the aqueous phase and it is mediated by colipase and a micelle. We believe that, in vivo, the formation of a complex between inactive PL, CL and a mixed micelle (Fig. 2) activates the enzyme by stabilizing the open conformation and exposing a large hydrophobic surface. This surface should facilitate complex binding to the underlying triglycerides of the emulsified duodenal oil particle.

Fig. 2. Schematic depiction of PL activation in solution by CL and a duodenal mixed micelle. The particle polar layer and underlaying substrates (not drawn to scale) are depicted in gray and yellow respectively.

Recently (Pignol, et al., 2000) (Abstract), [PDF], we have correlated the ability of different amphypathic compounds to activate PL, with their association with PC-PL in solution. Besides, the thecnique of small angle neutron scattering was used to characterize a solution containing PC-PL complex and taurodeoxycholate micelles. The resulting radius of gyration (56 A) and the match point of the solution indicate the formation of a ternary complex that is similar to the one observed in the neutron crystal structure (Figure 3). In addition, we show that either bile salts, lysophospholipids, or nonionic detergents that form micelles with radii of gyration ranging from 13 to 26 A are able to bind to the PC-PL complex, whereas smaller micelles or nonmicellar compounds are not. This further supports the notion of a micelle size-dependent affinity process for lipase activation in vivo.

Fig. 3. Different putative lipase-colipase-micelle associations in vivo. For each model, the open flap lipase es depicted in blue, colipase in cyan, and the bilesalt micelle as an orange circle. Models A and B are derived from the neutron crystal structure. In model C, the hydrophobic regions of the two activated lipase molecules are facing each other; in model D, they are directly interact with the bile salt micelle. The theoretical radius of gyration and match point percentages for each model are given. Only models A and B can explain the small angle neutron scattering experimental parameters (Rg= 55 A; match point, 39%).