Structure-function relationships in the carboxylic-ester-hydrolase superfamily. Disulfide bridge arrangement in porcine intestinal glycerol-ester hydrolase.
CNBr fragments from porcine intestinal glycerol-ester hydrolase were separated by SDS/PAGE under reducing and nonreducing conditions, and their amino-acid sequences were analysed. Two intra-chain disulfide bridges were identified, namely Cys70-Cys99 (loop A) and Cys256-Cys267 (loop B). As the Cys71 sulfhydryl group could not be alkylated with iodoacetamide, it is suggested that the residue is blocked rather than being present in the free form. The two disulfide bridges of intestinal glycerol-ester hydrolase are present in the cholinesterase family, although the enzyme showed only about 35% identity with these proteins. Furthermore, the finding that glycerol-ester hydrolase was partly inactivated under reducing conditions suggests that one or both disulfide bridges are important for the enzyme conformation. Lastly, glycerol-ester hydrolase was also found to hydrolyse cholinergic substrates, although residues Trp86 and Asp74 which are considered to be the main constituents of the 'anionic' subsite responsible for substrate binding in cholinesterases were absent from loop A. Other amino-acid residues in the glycerol-ester hydrolase may therefore be responsible for the binding of cholinergic substrates to the enzyme.
Smialowski-Fléter
S
S
Laboratoire de Biochimie et Biologie de la Nutrition, Faculté des Sciences et Techniques de St-Jérôme, Marseille, France.
Moulin
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A
Villard
C
C
Puigserver
A
A
D000818:Animals; D001665:Binding Sites; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry* / Q000378:metabolism; D002802:Cholinesterases / Q000737:chemistry; D003488:Cyanogen Bromide / Q000737:chemistry; D003545:Cysteine / Q000032:analysis; D004220:Disulfides / Q000737:chemistry*; D004229:Dithiothreitol / Q000494:pharmacology; D004795:Enzyme Stability; D007413:Intestinal Mucosa / Q000201:enzymology*; D007460:Iodoacetamide / Q000494:pharmacology; D008049:Lipase / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry; D010449:Peptide Mapping; D011485:Protein Binding; D011487:Protein Conformation; D017421:Sequence Analysis; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013552:Swine
D004220:Disulfides; D010446:Peptide Fragments; D002265:Carboxylic Ester Hydrolases; C034305:acylglycerol acylhydrolase; D008049:Lipase; D002802:Cholinesterases; D003545:Cysteine; D003488:Cyanogen Bromide; D004229:Dithiothreitol; D007460:Iodoacetamide