Abstract
Previous studies have postulated the presence of two bile salt-binding sites regulating the activity of the pancreatic bile salt-dependent lipase. One of these sites, located in an N-terminal basic cluster, has been identified as the specific bile salt-binding site. Interaction of primary bile salts with this proximal site induces the formation of a micellar binding site from a pre-existing nonspecific or pre-micellar bile salt-binding site. Here we have investigated the functional significance of another basic cluster comprised of amino acid residues Arg(423), Lys(429), Arg(454), Arg(458), and Lys(462), distal from the catalytic site. For this purpose these residues were mutagenized in Ile or Ala residues. The mutagenized enzyme lost activity on both soluble and emulsified substrates in the presence of bile salts. However, in the absence of bile salts, the mutagenized enzyme displayed the same activity on soluble substrate as the wild-type recombinant enzyme. Consequently, the distal basic cluster may represent the nonspecific (or pre-micellar) bile salt-binding site susceptible to accommodate primary and secondary bile salts. According to the literature, tyrosine residue(s) should participate in this site. Therefore, two tyrosine residues, Tyr(427) and Tyr(453), associated with the distal basic cluster were also mutagenized. Each tyrosine substitution to serine did not inhibit the enzyme activity on soluble substrate, independently of the presence of primary or secondary bile salts. However, the enzyme activity on cholesteryl oleate solubilized in primary bile salt micelles was decreased by mutations substantiating that these residues are part of the nonspecific bile salt-binding site.
Highlights
Bile salt-dependent lipase (BSDL,[1] EC 3.1.1.13), referred to as cholesteryl ester hydrolase or carboxyl ester lipase, is a lipolytic enzyme secreted by the acinar pancreatic cell into the duodenum where it plays a significant role in dietary lipid digestion
Expression of R423A/K429I/R454A/R458A/K462I Recombinant Mutagenized BSDL—The expression of BSDL bearing mutations of the distal basic cluster R423A, K429I, R454A, R458A, and K462I (R423A/K429I/R454A/R458A/K462I BSDL) was examined in all clones selected for G418 resistance and compared with that of the wild-type 3B clone (14) to that of the control clone
The conclusions from these studies indicated the possible presence of two bile saltbinding sites, regulating the enzyme activity both on water and lipid-soluble substrates
Summary
Bile salt-dependent lipase (BSDL,[1] EC 3.1.1.13), referred to as cholesteryl ester hydrolase or carboxyl ester lipase, is a lipolytic enzyme secreted by the acinar pancreatic cell into the duodenum where it plays a significant role in dietary lipid digestion. Binding of a monomeric primary bile salt to the specific site leads to the opening of an active site loop comprised of residues His[115] to Tyr[125] of the bovine BSDL, a loop that otherwise is in a closed conformation that might hinder substrate binding (6, 7). We have recently shown that this N-terminal basic cluster likely represents the socalled specific (or proximal) bile salt-binding site of BSDL susceptible to accommodating anionic ligands such as acidic phospholipids (11). This site may be involved in regulating the enzyme activity (11).
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