Uncoupling of catalysis and colipase binding in pancreatic lipase by limited proteolysis.
In the intestine, the hydrolysis of triglycerides by pancreatic lipase is performed only in the presence of colipase, whose function is to anchor lipase to the bile-salt-coated lipid interface. Biochemical and crystallographic data on porcine and human lipases have shown that the molecule is made of two well-delimited domains. In order to get more information on the role of the domains in catalysis and colipase binding, we performed limited proteolysis on lipase from various species and obtained different patterns of cleavage. In the case of porcine and human lipases, only the C-terminal domain (12 kDa) could be obtained after chymotryptic attack, whereas in the horse enzyme the cleavage of the Leu410-Thr411 bond gave rise to a large N-terminal (45 kDa) and a small C-terminal (4 kDa) fragment. The isolated porcine and human C-terminal domains were completely inactive towards emulsified tributyrin, though were able to bind colipase. Conversely, the horse 45 kDa fragment retained the lipase activity but failed to correctly bind colipase. This work definitely proves that catalysis and colipase binding are separate events involving topographically distinct regions of the molecule and focuses attention on the role of the C-terminal domain in colipase binding.
Abousalham
A
A
Centre de Biochimie et de Biologie Moléculaire du Centre National de la Recherche Scientifique, Marseille, France.
Chaillan
C
C
Kerfelec
B
B
Foglizzo
E
E
Chapus
C
C
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002918:Chymotrypsin / Q000378:metabolism; D003089:Colipases / Q000378:metabolism; D006736:Horses; D006801:Humans; D008049:Lipase / Q000378:metabolism*; D008969:Molecular Sequence Data; D010179:Pancreas / Q000201:enzymology*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010449:Peptide Mapping; D011487:Protein Conformation; D013552:Swine; D014280:Triglycerides / Q000378:metabolism
D003089:Colipases; D010446:Peptide Fragments; D014280:Triglycerides; D008049:Lipase; D002918:Chymotrypsin; C005830:tributyrin