On the Probable Involvement of Arginine Residues in the Bile‐Salt‐Binding Site of Human Pancreatic Carboxylic Ester Hydrolase

Lagadic‐Gossmann, Dominique; Campése, Daniel; Multigner, Luc; Lafont, Huguette; De, Alain

doi:10.1111/j.1432-1033.1983.tb07466.x

Cited by 25 publications

(18 citation statements)

References 30 publications

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“…However, a bile-salt-mediated cross-binding of Tyr122 and Arg63 could induce a conformational change, especially as loop L,,,* (following Arg63) is probably not anchored by salt bridging in the carboxylester-lipase enzymes. The close vicinity and possible coupling of the non-specific site to the oxyanion hole loop (salmon carboxylester-lipase positions 105-113) may explain the increase in k,,, observed upon bile-salt-binding, especially with taurodeoxycholate [45].…”

Section: Hypothetical Bile-salt-binding Sitesmentioning

confidence: 99%

“…Binding of the hydroxy groups may be accomplished by Asnll7, Gln66 and Gln71, all carboxylester-lipase-conserved residues. However, niodelling studies showed that an involvement of the glutamine residues and Phe60 would require a different conformation of the shortened loop L,,,* and, to a certain extent, loop L:,+ A dimerization through this hypothetical site would bring the two non-specific sites together, and may explain the transformation of the non-specific site into a micellar and lipidbinding site [24,45,551. The possible bile-salt-binding mechanisms described in this study are summarized as a tentative model in Fig.…”

Section: Hypothetical Bile-salt-binding Sitesmentioning

confidence: 99%

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Pancreatic Carboxylester Lipase from Atlantic Salmon (Salmo salar)

Gjellesvik¹,

Lorens²,

Male³

1994

European Journal of Biochemistry

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We report the isolation and characterization of a 1795-bp cDNA fragment encoding Atlantic salmon pancreatic carboxylester lipase from salmon pancreas mRNA. The nearly full-length cDNA contained a 540-amino-acid open-reading frame, encompassing the mature protein (by similarity to mammalian carboxylester lipase enzymes). The salmon carboxylester lipase primary structure shared 58% identity with mammalian carboxylester lipases, lacking the proline-rich C-terminal repeats found in human and rat carboxylester lipases. Congruent with other esterase B type enzymes, the salmon carboxylester lipase contained a canonical serine-esterase catalytic triad motif consisting of serine, histidine and aspartic acid. Computer-assisted modelling of the tertiary structure for salmon carboxylester lipase was conducted using acetylcholine esterase (Torpedo californica) as a template structure. The model, in conjunction with sequence comparisons and available enzymological data, has been used to locate putative bile-salt-binding and lipid-binding sites. The carboxylester lipase enzymes contain a unique, highly conserved insert region that may be associated with bile-salt binding. In the model structure, this region is located close to the active site, and contains a tyrosine residue with an adjacent carboxylester-lipase-conserved arginine. These traits have previously been predicted for the non-specific (regarding bile-salt hydroxylation) bile-salt-binding site in carboxylester lipase enzymes. At this site, a dihydroxy or trihydroxy bile-salt molecule may bind the tyrosine via hydrophobic interactions, the anionic bile-salt head group may bind the arginine, while hydrogen bonding between the bile-salt 12u hydroxy group and an adjacent aspargine residue is possible. The model does not contain an active site 'lid' structure as found in other lipases. The carboxylester lipase structural homolog to the 'flap' of the lipases from Geotrichum candidum and Candida rugosa contains a carboxylester-lipase-conserved deletion that renders this region unable to cover the active site. Instead, the shortening of this loop leads to solvent exposure of the carboxylester lipase insert region, an additional indication of the functional importance of this region.Carboxylester lipase (also bile-salt-dependent lipase and cholesterol esterase) is one of the lipases secreted from the vertebrate pancreas [l, 21. In mammals, this enzyme is considered important for cholesterol ester and vitamin ester digestion [3], and in triacylglyceride digestion in conjunction with other digestive lipases [4, 51. The enzyme exhibits strict dependence on bile-salts for hydrolytic activity on insoluble lipid substrates.In teleosts, most studies conclude that the major, if not the only pancreatic lipolytic enzyme, is of the carboxylester lipase type [6-91. This was shown in cod (Gadus morhua), where the enzyme also has been purified and characterized Correspondence to D. R. Gjellesvik, WS Biotec-Mackzymal, Strandgata 3, N-9008 TromsG, NorwayAbbreviations. ClHgBzOH, p-chloromercu...

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Section: Hypothetical Bile-salt-binding Sitesmentioning

confidence: 99%

Section: Hypothetical Bile-salt-binding Sitesmentioning

confidence: 99%

Pancreatic Carboxylester Lipase from Atlantic Salmon (Salmo salar)

Gjellesvik¹,

Lorens²,

Male³

1994

European Journal of Biochemistry

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“…Early studies have proposed that bile salts interact with two sites on the protein (2). One site, specific for primary bile salts, is associated with enzyme dimerization and activation, whereas the second is less specific, is able to bind indistinctly primary and secondary bile salts, and is involved in the enzyme binding to micellar or aggregated substrates (3,4). More recently, the presence of these two bile salt-binding sites has been detected on the human milk counterpart enzyme referred to as bile salt-stimulated lipase (5).…”

mentioning

confidence: 99%

“…Binding of a monomeric primary bile salt to the specific site leads to the opening of a loop comprised of residues His 115 to Tyr 125 of the bovine BSDL, a loop that otherwise is in a closed conformation which might hinder substrate binding (6,7). Using chemical modification approaches, tyrosine and arginine have been identified as key residues for BSDL interaction with bile salts (3,4). Furthermore, a recent study demonstrated that Arg 63 is essential for the enzyme activity on substrates such as cholesteryl oleate solubilized by sodium taurocholate (9).…”

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confidence: 99%

Site-directed Mutagenesis of the Basic N-terminal Cluster of Pancreatic Bile Salt-dependent Lipase

Aubert¹,

Sbarra

Petit-Thévenin

et al. 2002

Journal of Biological Chemistry

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Previous studies have postulated the presence of a heparin-binding site on the bile salt-dependent lipase (BSDL), whereas two bile salt-binding sites regulate the enzyme activity. One of these sites may overlap with the tentative heparin-binding site at the level of an N-terminal basic cluster consisting of positive residues Lys 32 , Lys 56 , Lys 61 , Lys 62 , and Arg 63 . The present study uses specific site-directed mutagenesis to determine the functional significance of this basic cluster. Mutations in this sequence resulted in recombinant enzymes that were able to bind to immobilized and to cell-associated heparin before moving throughout intestinal cells. Recombinant BSDL was fully active on soluble substrate, but mutants were less active on micellar cholesteryl oleate in comparison with the wild-type enzyme. Activation studies by primary (sodium taurocholate) and by secondary (sodium taurodeoxycholate) bile salts revealed that the activation of BSDL by sodium taurocholate at concentrations below the critical micellar concentration, and not that evoked by micellar bile salts, was affected by substitutions, suggesting that this Nterminal basic cluster likely represents the specific bile salt-binding site of BSDL. Substitutions also affected the activation of the enzyme promoted by anionic phospholipids, extending the function of this site to that of a cationic regulatory site susceptible to accommodate anionic ligands.

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“…Early studies (2) have proposed that bile salts interact with two sites on the protein. One site, specific for primary bile salts, is associated with enzyme dimerization and activation, whereas the second site is less specific, able to bind indistinctly primary and secondary bile salts, and is involved in the enzyme binding to micellar or aggregated substrates (3,4). More recently the presence of these two bile salt-binding sites has been detected on the human milk counterpart enzyme referred to as bile salt-stimulated lipase (5).…”

mentioning

confidence: 97%

Site-directed Mutagenesis of the Distal Basic Cluster of Pancreatic Bile Salt-dependent Lipase

Aubert-Jousset¹,

Sbarra

Lagadic‐Gossmann

2004

Journal of Biological Chemistry

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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.

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On the Probable Involvement of Arginine Residues in the Bile‐Salt‐Binding Site of Human Pancreatic Carboxylic Ester Hydrolase

Cited by 25 publications

References 30 publications

Pancreatic Carboxylester Lipase from Atlantic Salmon (Salmo salar)

Pancreatic Carboxylester Lipase from Atlantic Salmon (Salmo salar)

Site-directed Mutagenesis of the Basic N-terminal Cluster of Pancreatic Bile Salt-dependent Lipase

Site-directed Mutagenesis of the Distal Basic Cluster of Pancreatic Bile Salt-dependent Lipase

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