Reactivation of the totally inactive pancreatic lipase RP1 by structure-predicted point mutations.
Both classical pancreatic lipase (DPL) and pancreatic lipase-related protein 1 (DPLRP1) have been found to be secreted by dog exocrine pancreas. These two proteins were purified to homogeneity from canine pancreatic juice and no significant catalytic activity was observed with dog PLRP1 on any of the substrates tested: di- and tri-glycerides, phospholipids, etc. DPLRP1 was crystallized and its structure solved by molecular replacement and refined at a resolution of 2.10 A. Its structure is similar to that of the classical PL structures in the absence of any inhibitors or micelles. The lid domain that controls the access to the active site was found to have a closed conformation. An amino-acid substitution (Ala 178 Val) in the DPLRP1 may result in a steric clash with one of the acyl chains observed in the structures of a C11 alkyl phosphonate inhibitor, a transition state analogue, bound to the classical PL. This substitution was suspected of being responsible for the absence of DPLRP1 activity. The presence of Val and Ala residues in positions 178 and 180, respectively, are characteristic of all the known PLRP1, whereas Ala and Pro residues are always present in the same positions in all the other members of the PL gene family. Introducing the double mutation Val 178 Ala and Ala 180 Pro into the human pancreatic 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.
Roussel
A
A
Architecture et Fonction des Macromolécules Biologiques, Marseille, France.
de Caro
J
J
Bezzine
S
S
Gastinel
L
L
de Caro
A
A
Carrière
F
F
Leydier
S
S
Verger
R
R
Cambillau
C
C
D000409:Alanine / Q000235:genetics; D000818:Animals; D004285:Dogs; D004789:Enzyme Activation; D006801:Humans; D007700:Kinetics; D008049:Lipase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010179:Pancreas / Q000201:enzymology*; D010189:Pancreatic Juice / Q000201:enzymology; D017354:Point Mutation; D011392:Proline / Q000235:genetics; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D014633:Valine / Q000235:genetics
D011392:Proline; D008049:Lipase; C075987:pancreatic lipase-related protein 1; D014633:Valine; D000409:Alanine