The single polypeptide chain of about 460 amino acids of porcine pancreatic lipase (EC 3.1.1.3) has been fragmented into five peptides by cyanogen bromide cleavage [Rovery, M., Bianchetta, J. Sr Guidoni, A. (1973) Biochim. Biophys. Acra, 328, 391 -3951. The sequence of the first three cyanogen bromide peptides (CNI, CNII, CNIII), including a total of 234 amino acids, was fully elucidated. Automatic or manual Edman degradation was performed on the different peptides. Fragmentations of the CN peptides were accomplished by digestions with trypsin (after citraconylation or 1,2-cyclohexanedione treatment), chymotrypsin and Staphylococcus aureus external protease. Hydrolysis of unreduced material by pepsin and thermolysin, performed in order to determine the S-S bridge positions, provided useful overlapping peptides.The glycan moiety of lipase is bound to Asn-166. The non-essential tyrosine specifically blocked by diisopropylphosphorofluoridate is Tyr-49 in a cluster of asparagine and glutamine residues. The existence of a highly hydrophobic sequence (206-217) at the C terminus of the CNII fragment is noteworthy.Pancreatic lipase promotes the partial hydrolysis of long-chain water-insoluble triglycerides by a typical heterogeneous catalysis [l-31. The most characteristic property of lipase [4-81, phospholipase [9,10] and probably other lipolytic enzymes is that of becoming more active by several orders of magnitude when the substrate passes from a monomeric to an aggregated or emulsified state. Therefore, lipase action involves three main steps, i.e. interfacial adsorption, interfacial activation and catalysis, for which suitable residues or groups of residues must exist in the enzyme molecule.Lipase is known to adsorb readily to hydrophobic interfaces such as those provided by natural triglyceride substrates [ll], siliconized glass beads in the presence [7] or absence 1121 of a substrate and monomolecular films of lipid spread on the surface of water [13]. On the other hand, it cannot adsorb to Miniprint Supplement. Some parts of this work, including Materials and Methods, the tables, Fig. S1-S4 and References are given in Miniprint at the end of the paper. interfaces which are hydrophilic by nature [12] or made hydrophilic by the accumulation of amphipathic molecules [14]. This latter case is of special importance ivl vivo because of the presence of large amounts of amphipathic bile salts in the duodenum during fat digestion. In the conditions in vivo, the inhibition of lipase is prevented by a small protein cofactor, colipase [15 -191, which adsorbs to hydrophilic interfaces and then serves as an anchor for lipase [12]. Therefore, in addition to the above-mentioned sites necessary for the catalytic cycle to proceed, lipase may be expected to have a special site recognizing adsorbed colipase in the presence of an interface. The elucidation of the primary structure of the enzyme represents a first attempt to identify and locate these sites involved in protein-lipid and protein-protein interactions.Specific react...
