Characterization of the serine reacting with diethyl p-nitrophenyl phosphate in porcine pancreatic lipase

Guidoni, A.; Benkouka, Fatima; De, Josiane; Rovery, M.

doi:10.1016/0005-2744(81)90120-0

Cited by 91 publications

(25 citation statements)

References 9 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…has been shown to inhibit pancreatic lipase irreversibly in the presence of colipase and micellar concentrations of NaTDC by reacting with the serine residue at the active site (52,53). This result indicates that the combined effect of NaTDC micelles and colipase is to open the lipase flap, thereby making the active site serine residue accessible to inhibition.…”

Section: Influence Of Nonionic Detergents and Colipase On Lipase Inhimentioning

confidence: 95%

Lipase Activation by Nonionic Detergents

Hermoso

Pignol

Kerfélec

et al. 1996

Journal of Biological Chemistry

205

View full text Add to dashboard Cite

The crystal structure of the ternary porcine lipasecolipase-tetra ethylene glycol monooctyl ether (TGME) complex has been determined at 2.8 Å resolution. The crystals belong to the cubic space group F23 with a ‫؍‬ 289.1 Å and display a strong pseudo-symmetry corresponding to a P23 lattice. Unexpectedly, the crystalline two-domain lipase is found in its open configuration. This indicates that in the presence of colipase, pure micelles of the nonionic detergent TGME are able to activate the enzyme; a process that includes the movement of an N-terminal domain loop (the flap). The effects of TGME and colipase have been confirmed by chemical modification of the active site serine residue using diisopropyl p-nitrophenylphosphate (E600). In addition, the presence of a TGME molecule tightly bound to the active site pocket shows that TGME acts as a substrate analog, thus possibly explaining the inhibitory effect of this nonionic detergent on emulsified substrate hydrolysis at submicellar concentrations. A comparison of the lipase-colipase interactions between our porcine complex and the human-porcine complex (van Tilbeurgh, H., Egloff, M.-P., Martinez, C., Rugani, N., Verger, R., and Cambillau, C. (1993) Nature 362, 814 -820) indicates that except for one salt bridge interaction, they are conserved. Analysis of the superimposed complexes shows a 5.4°rotation on the relative position of the N-terminal domains excepting the flap that moves in a concerted fashion with the C-terminal domain. This flexibility may be important for the binding of the complex to the water-lipid interface.Pancreatic lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) plays a key role in dietary fat absorption in the intestine. It converts insoluble long chain triglycerides into more polar molecules that are able to cross the brush border membrane of enterocytes as mixed micelles with bile salts. Besides their role in triglyceride digestion through lipid emulsification and intestinal fat absorption, bile salts exert a strong inhibitory effect by preventing lipase adsorption through coating of the water-lipid interface (1). Lipase adsorption is a fundamental process because the enzyme catalyzes a heterogeneous reaction that involves an interfacial activation step. To counteract the inhibitory effect of bile salts, the pancreas secretes a small protein, colipase (molecular mass, 10 kDa), which anchors lipase to the bile salt-coated water-lipid interface in their presence (2, 3). Thus, lipolysis results from the combined effect of pancreatic lipase, colipase, and bile salts.Pancreatic lipase is a single polypeptide chain of 50 kDa. This protein has been characterized in several species, and the amino acid sequences of the enzyme from pig, man, horse, rabbit, rat, guinea pig, and coypu (4 -10) have been reported. In addition, related lipases (type 1 and 2) have been found in dog, rat man, guinea pig, and coypu (11-16) by screening pancreatic cDNA libraries. The localization and role of these related lipases is not clear.Crystal structures of the human ...

show abstract

Section: Influence Of Nonionic Detergents and Colipase On Lipase Inhimentioning

confidence: 95%

Lipase Activation by Nonionic Detergents

Hermoso

Pignol

Kerfélec

et al. 1996

Journal of Biological Chemistry

205

View full text Add to dashboard Cite

show abstract

“…This is best exemplified by the difficulty in assigning a common active site serine among the family members. Early studies using emulsified diethyl p -nitrophenylphosphate to label PL identified a serine in a GXSXG pentapeptide sequence (where X ϭ any amino acid) as a residue involved in interfacial binding, but not the putative active site of the enzyme (31,32). Similar experiments of LPL treated with diisopropylfluorophosphate (DFP) yielded the conclusion that a labeled serine residue, in a GGS sequence, was the catalytic center of that enzyme (33).…”

Section: Lipase Gene Family Sequence Comparisonsmentioning

confidence: 99%

The lipase gene family

Wong

Schotz

2002

Journal of Lipid Research

265

192

View full text Add to dashboard Cite

“…The p-strand/ESer/a-helix structural motif including the Gly-X-Ser-X-Gly consensus sequence has been detected only in lipases and esterases (Derewenda & Cambillau, 1991;Derewenda & Sharp, 1993). The structure of HPL clearly demonstrated that Ser 152 is the nucleophilic residue essential for catalysis, in agreement with the chemical modification of Ser 152 in porcine pancreatic lipase (Guidoni et al 1981), but in contradiction to results suggesting a function of Ser 152 in interfacial recognition (Chapus & SCrCmiva, 1976). In the structure resolved by Winkler et al (1990), the active site is covered by a surface loop between the disulphide bridge Cys 237 and 261.…”

Section: E P I T O P E M a P P I N G Of H U M A N G A S T R I C L I Pmentioning

confidence: 55%