Action of organophosphates and sulfonyl halides on porcine pancreatic lipase

Maylié, M.F.; Charles, M.; Desnuelle, P.

doi:10.1016/0005-2744(72)90017-4

Cited by 85 publications

(34 citation statements)

References 30 publications

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“…For example, the enzymes lecithin-cholesterol acyltransferase and lipoprotein lipase are readily inactivated by DFP, but the modification occurs not on the catalytic serine but on a second serine that is probably related instead to the substrate-binding site (25). Several additional secreted lipases, most notably bacterial lipases from Staphylococcus hyicus (26) and E. coli (phospholipase A 2 ) (8) and pancreatic lipase (27,28), are insensitive to DFP and PMSF and yet use serine as the catalytic nucleophile. In the case of the two bacterial lipases, the involvement of a serine residue in the catalytic mechanism was inferred by their sensitivity to the long chain alkylating agent HDSF (8,29).…”

Section: Discussionmentioning

confidence: 99%

Structural Basis for the Insensitivity of a Serine Enzyme (Palmitoyl-Protein Thioesterase) to Phenylmethylsulfonyl Fluoride

Das¹,

Bellizzi²,

Tandel³

et al. 2000

Journal of Biological Chemistry

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Palmitoyl-protein thioesterase-1 (PPT1) is a newly described lysosomal enzyme that hydrolyzes long chain fatty acids from lipid-modified cysteine residues in proteins. Deficiency in this enzyme results in a severe neurodegenerative storage disorder, infantile neuronal ceroid lipofuscinosis. Although the primary structure of PPT1 contains a serine lipase consensus sequence, the enzyme is insensitive to commonly used serine-modifying reagents phenylmethylsulfonyl fluoride (PMSF) and diisopropylfluorophosphate. In the current paper, we show that the active site serine in PPT1 is modified by a substrate analog of PMSF, hexadecylsulfonylfluoride (HDSF) in a specific and site-directed manner. The apparent K i of the inhibition was 125 M (in the presence of 1.5 mM Triton X-100), and the catalytic rate constant for sulfonylation (k 2 ) was 3.3/min, a value similar to previously described sulfonylation reactions. PPT1 was crystallized after inactivation with HDSF, and the structure of the inactive form was determined to 2.4 Å resolution. The hexadecylsulfonyl was found to modify serine 115 and to snake through a narrow hydrophobic channel that would not accommodate an aromatic sulfonyl fluoride. Therefore, the geometry of the active site accounts for the reactivity of PPT1 with HDSF but not PMSF. These observations suggest a structural explanation as to why certain serine lipases are resistant to modification by commonly used serine-modifying reagents.Palmitoyl-protein thioesterase-1 (PPT1) 1 is a newly described lysosomal hydrolase that removes long chain fatty acids from lipid-modified cysteine residues in fatty acylated proteins (reviewed in Ref. 1). Deficiency of the enzyme leads to a lysosomal storage disease, infantile neuronal ceroid lipofuscinosis, which causes blindness, seizures, and cortical atrophy of the brain (2). Lysosomal inclusion bodies, termed granular osmiophilic deposits, accumulate in all tissues, and resemble lipofuscin deposits that occur during normal aging. In keeping with this important lipid-metabolizing role, PPT1 is one of the most abundant lysosomal enzymes in the brain (3). In contrast to most hydrolytic enzymes (such as proteases, lipases, esterases, and thioesterases), PPT1 is insensitive to the serine-modifying reagents phenylmethylsulfonyl fluoride (PMSF) and diisopropylfluorophosphate (DFP) (4). The insensitivity of PPT1 to these reagents was an important feature of PPT1 because it allowed for the identification of PPT activity in the presence of contaminating lipase and protease activities. We wondered whether there might be structural and/or mechanistic differences between PPT1 and these other lipolytic enzymes that would account for this observation. We have recently determined the three-dimensional crystal structure of PPT1 in the presence and absence of palmitoyl-CoA and shown that palmitate modifies serine 115 of the enzyme (5), confirming that a serine residue is the catalytic nucleophile.In the current study, we show that although PPT1 is insensitive to inactivation by the ser...

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Section: Discussionmentioning

confidence: 99%

Structural Basis for the Insensitivity of a Serine Enzyme (Palmitoyl-Protein Thioesterase) to Phenylmethylsulfonyl Fluoride

Das¹,

Bellizzi²,

Tandel³

et al. 2000

Journal of Biological Chemistry

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“…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

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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 ...

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“…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%