Fat digestion in humans and some mammals such as dogs requires the successive intervention of two lipases: gastric lipase, which is stable and active despite the highly acidic stomach environment, followed by the classical pancreatic lipase secreted into the duodenum. We previously solved the structure of recombinant human gastric lipase (HGL) at 3.0-Å resolution in its closed form; this was the first structure to be described within the mammalian acid lipase family. Among the mammalian lipases, the preduodenal and lysosomal lipases belong to a family of enzymes that are able to withstand acidic conditions, under which they continue to be active. This family shows no sequence homology with any other known lipase families. The preduodenal lipases form a group of closely related enzymes originating from the stomach, the tongue, or the pharynx, and none of them requires any specific protein cofactor for its activity (1-3).The state of malnutrition of patients suffering from exocrine pancreatic insufficiency, which is often associated with cystic fibrosis, can be improved by prescribing dietary supplements prepared from pancreatic extracts of animal origin. The use of gastric lipases may lead to considerable progress in the treatment of exocrine pancreatic insufficiency (4) because of their resistance to the gastric medium and their high lipolytic activity at the low pH values observed in the duodenum A screening program (1) and biochemical studies (5-7) led to the conclusion that dog gastric lipase (DGL), 1 a glycoprotein with a molecular mass of 49 kDa, was the most active enzyme when tested on long-chain triacylglycerols. These lipids are the main components of human dietary fats. The first attempts to produce an active recombinant DGL (r-DGL) in procaryotes (4) were unsuccessful. Joliff et al. (8) described the expression and secretion of an active r-DGL in the baculovirus insect cell system. However, with a view to therapeutic applications, r-DGL production by a safe eukaryotic system of expression such as transgenic plants provides an attractive alternative. Transgenic plants offer considerable advantages, such as large production yields and the fact that they entail no risk of contamination by pathogens infectious to humans, whereas they contain the cell machinery mediating eukaryotic protein modifications. The first transgenic plants producing r-DGL were constructed in tobacco (9) and then in maize by Meristem therapeutics in collaboration with the Institut de Recherche Jouveinal/Parke-Davis during the last decade.All the triglyceride lipases for which the three-dimensional structures have been determined so far belong to the serine esterase class (10 -14). The active serine is part of a catalytic triad, which is similar to that observed in the case of serine proteases. The three-dimensional structure of the Candida rugosa lipase, which is inhibited by two enantiomers of a phosphonate inhibitor, provided the structural basis for establishing the chiral preferences of lipases (15). Covalent adducts of Rhizomu...

The crystal structures of gastric lipases in the apo form [Roussel, A., et al. (1999) J. Biol. Chem. 274, 16995-17002] or in complex with the (R(P))-undecyl butyl phosphonate [C(11)Y(4)(+)] [Roussel, A., et al. (2002) J. Biol. Chem. 277, 2266-2274] have improved our understanding of the structure-activity relationships of acid lipases. In this report, we have performed a kinetic study with dog and human gastric lipases (DGL and HGL, respectively) using several phosphonate inhibitors by varying the absolute configuration of the phosphorus atom and the chain length of the alkyl/alkoxy substituents. Using the two previously determined structures and that of a new crystal structure obtained with the other (S(P))-phosphonate enantiomer [C(11)Y(4)(-)], we constructed models of phosphonate inhibitors fitting into the active site crevices of DGL and HGL. All inhibitors with a chain length of fewer than 12 carbon atoms were found to be completely buried in the catalytic crevice, whereas longer alkyl/alkoxy chains were found to point out of the cavity. The main stereospecific determinant explaining the stronger inhibition of the S(P) enantiomers is the presence of a hydrogen bond involving the catalytic histidine as found in the DGL-C(11)Y(4)(-) complex. On the basis of these results, we have built a model of the first tetrahedral intermediate corresponding to the tristearoyl-lipase complex. The triglyceride molecule completely fills the active site crevice of DGL, in contrast with what is observed with other lipases such as pancreatic lipases which have a shallower and narrower active site. For substrate hydrolysis, the supply of water molecules to the active site might be achieved through a lateral channel identified in the protein core.

No abstract