Although structurally similar to pancreatic lipase (PL), the key enzyme of intestinal fat digestion, pancreatic lipase-related protein type 2 (PLRP2) differs from PL in certain functional properties. Notably, PLRP2 has a broader substrate specificity than PL, and unlike that of PL, its activity is not restored by colipase in the presence of bile salts. In the studies presented here, the activation mechanism of horse PLRP2 was studied through active site-directed inhibition experiments, and the results demonstrate fundamental differences with that of PL. The opening of the horse PLRP2 flap occurs as soon as bile salt monomers are present, is accelerated in the presence of micelles, and does not require the presence of colipase. Moreover, in contrast to PL, horse PLRP2 is able to directly interact with a bile salt micelle to form an active binary complex, without the micelle being presented by colipase, as evidenced by molecular sieving experiments. These findings, together with the sensitivity of the horse PLRP2 flap to partial proteolysis, are indicative of a higher flexibility of the flap of horse PLRP2 relative to PL. From these results, it can be concluded that PLRP2 can adopt an active conformation in the intestine, which could be important for the further understanding of the physiological role of PLRP2. Finally, this work emphasizes the essential role of colipase in lipase catalysis at the lipid-water interface in the presence of bile.

The organization of the pancreatic lipase in two well defined domains has been correlated to a specific function for each domain, catalytic activity for the N-terminal domain and colipase binding for the C-terminal domain. In order to see if such an organization implies that the two domains can behave as separate entities, we expressed the N- and C-terminal domains in insect cells. The recombinant proteins secreted in the cell supernatants present the expected molecular properties. However, whereas the C-terminal domain retains its function of colipase binding, the N-terminal domain appears to be unable to ensure catalysis. The lack of activity of the recombinant N-terminal domain could result either from a (partially) incorrect folding or from an incapacity to function by itself. These results suggest that, although both are structurally well defined, the two domains of the pancreatic lipase behave differently when they are expressed as separate entities.

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