Partial deletion of β9 loop in pancreatic lipase-related protein 2 reduces enzyme activity with a larger effect on long acyl chain substrates

Structural studies on pancreatic lipase have revealed a complex architecture of surface loops surrounding the enzyme active site and potentially involved in interactions with lipids. Two of them, the lid and beta 9 loop, expose a large hydrophobic surface and are considered as acyl chain binding sites based on their interaction with an alkyl phosphonate inhibitor. While the role of the lid in substrate recognition and selectivity has been extensively studied, the implication of beta 9 loop in acyl chain stabilization remained hypothetical. The characterization of an enzyme with a natural deletion of the lid, guinea pig pancreatic lipase-related protein 2 (GPLRP2), suggests however an essential contribution of the beta 9 loop in the stabilization of the acyl enzyme intermediate formed during the lipolysis reaction. A GPLRP2 mutant with a seven-residue deletion of beta 9 loop (GPLRP2-Delta beta 9) was produced and its enzyme activity was measured using various substrates (triglycerides, monoglycerides, galactolipids, phospholipids, vinyl esters) with short, medium and long acyl chains. Whatever the substrate tested, GPLRP2-,6439 activity is drastically reduced compared to that of wild-type GPLRP2 and this effect is more pronounced as the length of substrate acyl chain increases. Changes in relative substrate selectivity and stereoselectivity remained however weak. The deletion within 39 loop has also a negative effect on the rate of enzyme inhibition by alkyl phosphonates. All these findings indicate that the reduced enzyme turnover observed with GPLRP2-Delta beta 9 results from a weaker stabilization of the acyl enzyme intermediate due to a loss of hydrophobic interactions. (C) 2013 Elsevier B.V. All rights reserved.