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-related protein 1 (DPLRP1) have been found to be secreted by the exocrine pancreas. These two proteins were purified to homogeneity from canine pancreatic juice and no significant catalytic activity was observed with DPLRP1 on any of the substrates tested: di- and tri-glycerides; phospholipids (PC); 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 pancreatic lipase (PL) structures determined 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 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 inhibitors, bound to the classical PL. The presence of Val and Ala residues in positions 178 and 180, respectively, are characteristic of the three known pancreatic lipase-related protein 1 (PLRP1), whereas Ala and Pro residues are always present at 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-related protein 1 (HPLRP1) gene yielded a well expressed and folded enzyme in insect cells. This enzyme is kinetically active on tributyrin (1800 U/mg) as well as trioctanoin (2250 U/mg) and its activity is low in the presence of taurodeoxycholate and stimulated in the presence of colipase. Our findings on DPLRP1 and HPLRP1 are therefore likely to apply to all the PLRP1 lipases.
Bezzine
S
S
Architecture et Fonction des Macromolécules Biologiques, CNRS-IFR1 UPR9039, Marseille, France.
Roussel
A
A
de Caro
J
J
Gastinel
L
L
de Caro
A
A
Carrière
F
F
Leydier
S
S
Verger
R
R
Cambillau
C
C
D000595:Amino Acid Sequence; D000818:Animals; D018360:Crystallography, X-Ray; D004285:Dogs; D004789:Enzyme Activation; D007700:Kinetics; D008049:Lipase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D010189:Pancreatic Juice / Q000737:chemistry / Q000201:enzymology; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D014280:Triglycerides / Q000378:metabolism*
D014280:Triglycerides; D008049:Lipase; C075987:pancreatic lipase-related protein 1