The cysteine residues of recombinant human gastric lipase.
Recombinant human gastric lipase (rHGL) and three of its cysteine mutants (cysteine 227, 236, and 244 substitued for threonine or serine) were expressed in the baculovirus/insect cell system and purified to homogeneity by performing a two-step procedure. Substituting Ser for Cys 227 and Cys 236 resulted in mutant lipases with a significantly lower level of activity (30% and 22%, respectively) on a short chain triglyceride (tribuyrin) substrate, while the mutation at position 244 only slightly reduced the activity. Using 4, 4'-dithiopyridine (4-PDS) as a sulfhydryl reagent on the above mutants, it was possible to clearly identify the single sulfhydryl residue at position 244 and consequently, the disulfide bridge at position 227-236. No potential disulfide bridges were formed during the protein folding between cysteines 227-244 or between cysteines 236-244, as thought to occur in the case of rabbit gastric lipase (RGL). The present results are consistent with the recently determined 3D-structure of rHGL.
Canaan
S
S
Laboratoire de Lipolyse Enzymatique, UPR 9025 de l'IFR-1 du CNRS, 31 chemin Joseph Aiguier, Marseille Cedex 20, 13402, France. canaan@ibsm.cnrs-mrs.fr
Rivière
M
M
Verger
R
R
Dupuis
L
L
D019943:Amino Acid Substitution; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002384:Catalysis / Q000187:drug effects; D002460:Cell Line; D003545:Cysteine / Q000235:genetics / Q000378:metabolism*; D004220:Disulfides / Q000378:metabolism / Q000494:pharmacology; D005753:Gastric Mucosa / Q000201:enzymology*; D006801:Humans; D007313:Insecta; D007700:Kinetics; D008049:Lipase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017510:Protein Folding; D011725:Pyridines / Q000378:metabolism / Q000494:pharmacology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D017186:Titrimetry; D014280:Triglycerides / Q000378:metabolism
D004220:Disulfides; D011725:Pyridines; D011994:Recombinant Proteins; D014280:Triglycerides; C007542:4,4'-dipyridyl disulfide; D012967:Sodium Dodecyl Sulfate; C042861:4-thiopyridine; D008049:Lipase; D003545:Cysteine; C005830:tributyrin