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 separa ted by SDS/PAGE under reducing and nonreducing conditions, and their amino- acid sequences were analysed. Two intra-chain disulfide bridges were identi fied, 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. Th e two disulfide bridges of intestinal glycerol-ester hydrolase are present in the cholinesterase family, although the enzyme showed only about 35% ide ntity with these proteins. Furthermore, the finding that glycerol-ester hyd rolase was partly inactivated under reducing conditions suggests that one o r both disulfide bridges are important for the enzyme conformation. Lastly, glycerol-ester hydrolase was also found to hydrolyse cholinergic substrate s, although residues Trp86 and Asp74 which are considered to be the main co nstituents of the 'anionic' subsite responsible for substrate binding in ch olinesterases were absent from loop A. Other amino-acid residues in the gly cerol-ester hydrolase may therefore be responsible for the binding of choli nergic substrates to the enzyme.