title: The cysteine residues of recombinant human gastric lipase.
abstract: 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.
authors: 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
authors: S
authors: S
authors: Canaan
authors: M
authors: M
authors: Rivière
authors: R
authors: R
authors: Verger
authors: L
authors: L
authors: Dupuis
mesh_terms: 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
chemical_list: 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