AN INACTIVE PANCREATIC LIPASE-RELATED PROTEIN IS ACTIVATED INTO A TRIGLYCERIDE-LIPASE BY MUTAGENESIS BASED ON THE 3-D STRUCTURE

Both classical dog pancreatic lipase (DPL) and dog pancreatic lipase-r elated protein 1 (DPLRP1) have been found to be secreted by the exocri ne pancreas. These two proteins were purified to homogeneity from cani ne pancreatic juice and no significant catalytic activity was observed with DPLRP1 on any of the substrates tested: di-and tri-glycerides; p hospholipids (PC); etc. DPLRP1 was crystallized and its structure solv ed by molecular replacement and refined at a resolution of 2.10 Angstr om. Its structure is similar to that of the classical pancreatic lipas e (PL) structures determined in the absence of any inhibitors or micel les. The lid domain that controls the access to the active site was fo und to have a closed conformation. An amino-acid substitution (Ala 178 Val) in the DPLRP1 was suspected of being responsible for the absence of enzymatic activity by inducing a steric clash with one of the acyl chain observed in the structures of chiral C11 alkyl phosphonate inhi bitors, bound to the classical FL. The presence of Val and Ala residue s in positions 178 and 180, respectively, are characteristic of the th ree known pancreatic lipase-related protein 1 (PLRP1), whereas Ala and Pro residues are always present at the same positions in all the othe r members of the PL gene family. Introducing the double mutation Val 1 78 Ala and Ala 180 Pro into the human pancreatic-related protein 1 (HP LRP1) gene yielded a well expressed and folded enzyme in insect cells. This enzyme is kinetically active on tributyrin (1800 U/mg) as well a s trioctanoin (2250 U/mg) and its activity is low in the presence of t aurodeoxycholate and stimulated in the presence of colipase. Our findi ngs on DPLRP1 and HPLRP1 are therefore likely to apply to all the PLRP 1 lipases. (C) 1998 Elsevier Science Ireland Ltd. All rights reserved.