Adsorption of protein molecules at the lipid-water interface often leads to unfolding and denaturation. For pancreatic lipases, adsorption at the interface results in decreased activity and consequently inactiva-tion. In the physiological environment, the activity of pancreatic lipases is regulated by the presence of bile salts (cholic and chenodeoxycholic acids conjugated with glycine or taurine, such as taurodeoxycholate, glycodeoxycholate, or taurocholate) and other amphi-philic compounds, such as phospholipids and proteins. The presence of bile salts creates a negatively charged film on the triacylglycerol micelle that prevents lipase adsorption (32, 33). The cooperative formation of an enzyme-bile salt complex with a dissociation constant of 1.4 prevents the adsorption to the inter face (34). For a pancreatic lipase to act at the interface, the enzyme requires the binding of colipase, a 10-kDa protein also secreted by the exocrine pancreas, to facilitate adsorption of the enzyme at bile salt-coated lipid-

water interface. The inhibitory action of bile salts is observed on both microbial and pancreatic lipases, but the activity of the pancreatic enzyme can only be restored by the addition of pancreatic colipase, indicating that specific interactions occur between the pancreatic lipase and colipase (35).

Porcine pancreatic colipase is a single polypeptide chain containing 96 amino acid residues with five dis-ulfide bridges, and has an isoelectric point of 5.0 (36). Its primary structure has been determined by protein sequencing (37, 38). The protein is synthesized as a procolipase of 101 amino acid residues, and the active form, colipase96, is produced by proteolytic (tryptic) removal of the N-terminal pentapeptide (Val-Pro-Asp-Pro-Arg) (39, 40).

Colipase consists of a small N-terminal region and a three-fingers region held together by disulfide bridges (41, 42). Colipase exhibits an overall amphipathic character with most hydrophilic residues found in the N-terminal region, whereas most of the hydrophobic residues are located at the tip of the fingers in the opposite side that constitute the lipid-binding site (43-46).

Binding of colipase to lipase occurs between two hairpin loops (residues 44-46, 65-67) in the N-terminal region of colipase, and the ft-sheet of the C-terminal domain of the lipase molecule (41, 46). The interactions are mostly hydrophilic with the formation of two salt bridges and six hydrogen bonds. Porcine pancreatic lipase binds to colipase to form a 1:1 complex with a Kd of 5 x in the absence of an interface

(34, 47, 48). In the presence of 2 mM sodium tauro-

deoxycholate and 150 mM NaCl, binding of lipase to colipase at pH 7.0 has a Kd of 2 x 10~6 M (49).

The binding of colipase to the bile salt-liquid substrate at the interface involves the three-finger region that forms a hydrophobic surface interacting with the lipid (Fig. 3) (41). The central finger consists of three highly conserved Tyr residues (Tyr55, Tyr58, Tyr59 in porcine prolipase) in a strongly hydrophobic loop (also designated as the tyrosine loop region) that has long been implicated as the lipid-binding site (50-52). The binding of colipase to the substrate, tributyrin, at pH 7.0 in the presence of 4 mM sodium taurodeoxycholate and 150 mM NaCl has a dissociation constant Kd = 3.3 x 10~7 M (53). The binding of colipase to an emulsion of long-chain triacylglycerols stabilized with

Figure 3 The structure of human pancreatic lipase-colipase complex and its comformational change in the presence of mixed phospholipid/bile salt micelles. (Reprinted from Riddihough 1993, Nature 362, 793, with permission. Copyright 1993 Macmillan Magazine, Ltd.)

phosphatidylcholine and glycerol showed a Kd of 1.2 x in the presence of 4 mM sodium taurodeoxy-cholate and 150 mM NaCl at pH 7.0.

Colipase also binds to the surface helix (the lid) covering the catalytic triad of lipase in its open conformation (54, 55). The interaction is mediated by three hydrogen bonds between Arg38, Leu16, and Glu15 of colipase, and Val246, Ser343, and Asn240 of the lid. The binding serves to stabilize the lid during activation, and provides an extended hydrophobic surface that constitutes the lipid-water interface binding site, which is more than 50 A in length and has a surface area of approximately 965A2 (42, 56).

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