REACTIVATION OF THE TOTALLY INACTIVE PANCREATIC LIPASE RP1 BY STRUCTURE-PREDICTED POINT MUTATIONS

Both classical pancreatic lipase (DPL) and pancreatic lipase-related p rotein 1 (DPLRP1) have been found to be secreted by dog exocrine pancr eas. These two proteins were purified to homogeneity from canine pancr eatic juice and no significant catalytic activity was observed with do g PLRP1 on any of the substrates tested: di- and tri-glycerides, phosp holipids, etc. DPLRP1 was crystallized and its structure solved by mol ecular replacement and refined at a resolution of 2.10 Angstrom Its st ructure is similar to that of the classical PL structures in the absen ce of any inhibitors or micelles, The lid domain that controls the acc ess to the active site was found to have a closed conformation. An ami no-acid substitution (Ala 178 Val) in the DPLRP1 may result in a steri c clash with one of the acyl chains observed in the structures of a C1 1 alkyl phosphonate inhibitor, a transition state analogue, bound to t he classical FL. This substitution was suspected of being responsible for the absence of DPLRP1 activity. The presence of Val and Ala residu es in positions 178 and 180, respectively, are characteristic of all t he known PLRP1, whereas Ala and Pro residues are always present in the same positions in all the other members of the PL gene family. Introd ucing the double mutation Val 178 Ala and Ala 180 Pro into the human p ancreatic RP1 (HPLRP1) gene yielded a well expressed and folded enzyme in insect cells. This enzyme is kinetically active on triglycerides, Our findings on DPLRP1 and HPLRP1 are therefore likely to apply to all the RP1 Lipases. (C) 1998 Wiley-Liss, Inc.