The crystal structure of the ternary porcine lipasecolipase-tetra ethylene glycol monooctyl ether (TGME) complex has been determined at 2.8 Å resolution. The crystals belong to the cubic space group F23 with a ‫؍‬ 289.1 Å and display a strong pseudo-symmetry corresponding to a P23 lattice. Unexpectedly, the crystalline two-domain lipase is found in its open configuration. This indicates that in the presence of colipase, pure micelles of the nonionic detergent TGME are able to activate the enzyme; a process that includes the movement of an N-terminal domain loop (the flap). The effects of TGME and colipase have been confirmed by chemical modification of the active site serine residue using diisopropyl p-nitrophenylphosphate (E600). In addition, the presence of a TGME molecule tightly bound to the active site pocket shows that TGME acts as a substrate analog, thus possibly explaining the inhibitory effect of this nonionic detergent on emulsified substrate hydrolysis at submicellar concentrations. A comparison of the lipase-colipase interactions between our porcine complex and the human-porcine complex (van Tilbeurgh, H., Egloff, M.-P., Martinez, C., Rugani, N., Verger, R., and Cambillau, C. (1993) Nature 362, 814 -820) indicates that except for one salt bridge interaction, they are conserved. Analysis of the superimposed complexes shows a 5.4°rotation on the relative position of the N-terminal domains excepting the flap that moves in a concerted fashion with the C-terminal domain. This flexibility may be important for the binding of the complex to the water-lipid interface.Pancreatic lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) plays a key role in dietary fat absorption in the intestine. It converts insoluble long chain triglycerides into more polar molecules that are able to cross the brush border membrane of enterocytes as mixed micelles with bile salts. Besides their role in triglyceride digestion through lipid emulsification and intestinal fat absorption, bile salts exert a strong inhibitory effect by preventing lipase adsorption through coating of the water-lipid interface (1). Lipase adsorption is a fundamental process because the enzyme catalyzes a heterogeneous reaction that involves an interfacial activation step. To counteract the inhibitory effect of bile salts, the pancreas secretes a small protein, colipase (molecular mass, 10 kDa), which anchors lipase to the bile salt-coated water-lipid interface in their presence (2, 3). Thus, lipolysis results from the combined effect of pancreatic lipase, colipase, and bile salts.Pancreatic lipase is a single polypeptide chain of 50 kDa. This protein has been characterized in several species, and the amino acid sequences of the enzyme from pig, man, horse, rabbit, rat, guinea pig, and coypu (4 -10) have been reported. In addition, related lipases (type 1 and 2) have been found in dog, rat man, guinea pig, and coypu (11-16) by screening pancreatic cDNA libraries. The localization and role of these related lipases is not clear.Crystal structures of the human ...

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. Here, we present the neutron structure of the activated lipase-colipase-micelle complex as determined using the D2O/H2O contrast variation low resolution diffraction method. In the ternary complex, the disk-shaped micelle interacts extensively with the concave face of colipase and the distal tip of the C-terminal domain of lipase. 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 is mediated by colipase and a micelle.

No abstract