Competitive inhibition of lipolytic enzymes. I. A kinetic model applicable to water-insoluble competitive inhibitors

Ransac, Stéphane; Rivière, C.; Soulie, Jean-Michel; Gancet, C.; Verger, Robert; Haas, G.H. de

doi:10.1016/0005-2760(90)90110-j

Cited by 86 publications

(60 citation statements)

References 17 publications

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“…The inhibitory power (2) of the (R)-2-acylamino substrate analogues was determined as described by De Haas et al (1990 b) and by Ransac et al (1990). The substrate used in these studies was Lau,GroPCho.…”

Section: Plaz Assays and Inhibition Of Pla Activity By Substrate Anamentioning

confidence: 99%

“…Plotting R, as a function of the mole fraction inhibitor a yields a straight line, the slope of which yields the value of Z. For mixed micelles Ransac et al (1990) published a formula describing the relation between Z and the two-dimensional concentrations and binding constants of detergent, substrate and inhibitors. In the present study we use sodium taurodeoxycholate as a detergent which has been shown to have little or no affinity for the active site of porcine pancreatic PLA, .…”

Section: Plaz Assays and Inhibition Of Pla Activity By Substrate Anamentioning

confidence: 99%

“…Model representing the action of a lipolytic enzyme at the lipid-water interface. This model is a slightly modified version of the one published by Ransac et al (1990). E* represents the enzyme interacting with the interface, E*S and E*I the interfacial substrateenzyme and inhibitor-enzyme complexes, respectively.…”

Section: Direct Binding Studies Of Inhibitors To the Active Site Of Plamentioning

confidence: 99%

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Arginine 53 is involved in head‐group specificity of the active site of porcine pancreatic phospholipase A₂

Lugtigheid

Otten-Kuipers

Verheij

et al. 1993

European Journal of Biochemistry

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The X-ray structure of a mutant porcine pancreatic phospholipase A, inhibitor complex [Thunnissen et al. (1990) Nature 347, 689-6911 has been determined. This structure shows several interactions between the sn-2-acyl chain and the phosphate moiety of the inhibitor at sn-3 and the protein. The interactions of the remaining part of the polar head group are less clear. Because Arg53 is in close proximity to the head group, we tested the importance of charge at position 53 on enzymatic activity and specificity. Arg53 has been replaced by a glutamine and a glutamic acid in mutants R53Q and R53E, respectively. The effects of the mutations were tested with both zwitterionic and anionic substrates. With monomeric, zwitterionic, (R,S)-1,2-dihexanoyldithiopropyl-3-phosphocholine as substrate, the mutants R53Q and R53E display twofold and sevenfold, respectively, increased kJKm values, composed of increased k,, and decreased K , values. Tested on micelles of zwitterionic (R)-l,2-dioctanoylglycero-3-phosphocholine the mutants R53Q and R53E are more active than the native enzyme, whereas these mutations have an opposite effect on the activity on anionic (R)-l,2-dioctanoylglycero-3-phosphoglycol. Thus, whereas the native enzyme is 0.3 times as active on zwitterionic as on the anionic substrate, these ratios are 1.0 (R53Q) and 1.7 (R53E) for the mutants. No changes in activity were observed with the anionic substrate (R)-1,2-dioctanoylglycero-3-sulfate. Binding studies with substrate-derived inhibitors confirmed the increased affinity for zwitterionic phospholipids and the reduced affinity for anionic phospholipids. The kinetic and binding data indicate the involvement of the charge of residue 53 in head-group specificity and suggest a position of residue 53 closer to the choline or glycol than to the phosphate.The lipolytic enzyme phospholipase A, (PLA,) hydrolyzes the sn-2 ester bond of phosphoglycerides in a calcium-dependent and stereospecific reaction. Phospholipases occur both extracellularly and intracellularly. The extracellular enzymes are found abundantly in mammalian pancreas and in snake or bee venoms serving a digestive function. In addition the venom phospholipases show a wide range of pharmacological actions. The intracellular phospholipases are present in low concentrations in almost every mammalian cell and have an important role in inflammation processes by Abbreviations. PLA,, phospholipase A,; R53Q and R53E, mutants of PLA, with Arg53 changed to glutamine and glutamic acid, respectively ; Oco,GroSO,H, (R)-l,2-dioctanoylglycero-3-sulfate ; Oco,GroPGlo, (R)-l,2-dioctanoylglycero-3-phosphoglycol; Oc0,GroPCho, (R)-l,2-dioctanoylglycero-3-phosphocholine; Lau,GroPCho, (R)-l,2-didodecanoylglycero-3-phosphocholine; HxoS,GroPCho, (R,S')-1,2dihexanoyldithiopropyl-3-phosphocholine; DodPCho, n-dodecylphosphocholine; HxdPCho, n-hexadecylphosphocholine; MyrAholPCho, (R)-2-tetradecanoylaminohexanol-l-phosphocholine ; MyrAholPGlo, (R)-2-tetradecanoylaminohexanol-1 -phosphoglycol; KL, two-dimensional Michaelis Menten const...

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Section: Plaz Assays and Inhibition Of Pla Activity By Substrate Anamentioning

confidence: 99%

Section: Plaz Assays and Inhibition Of Pla Activity By Substrate Anamentioning

confidence: 99%

Section: Direct Binding Studies Of Inhibitors To the Active Site Of Plamentioning

confidence: 99%

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Arginine 53 is involved in head‐group specificity of the active site of porcine pancreatic phospholipase A₂

Lugtigheid

Otten-Kuipers

Verheij

et al. 1993

European Journal of Biochemistry

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“…The reservoir compartment was 148 mm wide and 249 mm long, Mixed films were spread from a chloroform solution (about 2 mg/ ml), over the surface of the reaction compartment only, whereas the reservoir was covered with a film of pure substrate (dicaprin) [22, 231. The kinetic data were analyzed as in the case of a previous model [14,241.…”

Section: Enzyme Kinetic Experimentsmentioning

confidence: 99%

“…In this model, we only consider cases where the regeneration of enzyme form, E*, from ET is very slow, and therefore does not take place during our experimental procedure. The kinetic treatment of this model is derived from the analogous treatment described by Ransac et al [14]. In the present kinetic treatment, the inhibitor is assumed to be in large molar excess compared to the adsorbed enzyme, giving the following sigmoidal expression of the time dependence of the products released:…”

Section: Kinetic Ntodel Illustrating Rhe Covalent Inactivation Of a Lmentioning

confidence: 99%

Inactivation of pancreatic and gastric lipases by tetrahydrolipstatin and alkyl‐dithio‐5‐(2‐nitrobenzoic acid)

Ransac

Gargouri

Moreau

et al. 1991

European Journal of Biochemistry

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We studied the covalent inhibition of lipases by the monolayer technique. We report the inactivation of porcine pancreatic and human and rabbit gastric lipases, acting on mixed monomolecular films of dicaprin containing tetrahydrolipstatin or new hydrophobic disulfide compounds, which can be described as a 'poisoned-interface' system.A kinetic model is presented for depicting the covalent inactivation of lipolytic enzymes at a lipid/water interface. The stoichiometry of the interfacial situation can be described as follows: one lipase molecule embedded among lo5 substrate molecules will be inactivated to half its initial velocity by the presence of 10 tetrahydrolipstatin molecules. This inactivation was independent of the surface pressure.When tested in the form of mixed films, all the disulfide compounds investigated specifically reduced the hydrolysis of 1,2-didecanoyl-sn-glycerol films by gastric lipases, but did not affect hydrolysis by pancreatic lipase. With this poisoned-interface system, tetrahydrolipstatin was found to be the most potent inactivator, whereas disulfide compounds showed a higher degree of selectivity than tetrahydrolipstatin.Gastric lipase differs from pancreatic lipase in several major respects. Under acidic pH conditions, gastric lipase have been found to be remarkably stable and active, whereas pancreatic lipase irreversibly loses its lipolytic capacity. The optimum pH for gastric lipase activity is around 5.4 [l, 21, compared to 8-9 in.the case of pancreatic lipase [3]. The partial hydrolysis of alimentary triacylglycerols which occurs at the pH levels prevailing in the stomach rapidly triggers pancreatic lipase activity in the intestine [4].We have shown that some amphiphiles (bile salts, alimentary proteins and phospholipids), which inhibit pure PPL [3], activate gastric lipase, since they can prevent the irreversible denaturation of the latter enzymes at substrate/water interfaces [l, S].Human gastric lipase (HGL) and rabbit gastric lipase (RGL) are enzymes which have been found to be inactivated by classical sulfhydryl reagents [5,S'-dithiobis(2-nitrobenzoic acid), NbS, ; 4,4'-dipyridyl disulfide) and by a hydrophobic disulfide compound: dodecyl-dithio-S-(2-nitrobenzoic acid) (12: 0-S-SNb) [6, 71. More recently, we reported that ajoene, derived from ethanol extracts of garlic, specifically inactivated HGL and RGL and had no effect on porcine pancreatic lipase With emulsified systems, it is not possible to control the 'interfacial quality' and to easily assess the distribution of soluble versus adsorbed amphiphilic molecules. This prompted us to use the monolayer technique, based upon surface-pressure decrease due to lipid-film hydrolysis [16 -181. This technique is applicable to those cases where the lipid forms a stable monomolecular film at the air/water interface and where reaction products are freely soluble and diffuse away rapidly into the aqueous phase.We studied, for the first time, the covalent inhibition of lipases by the monolayer technique. We report the inactivati...

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Synthesis and properties of novel lipopeptides and lipid mimetics

et al. 1997

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Lipid mimetics, synthetic molecules that resemble natural lipids either structurally or functionally, have been developed as potential medicinal substances. They have been successfully applied in the development of drug and peptide delivery systems and for the development of inhibitors or lipid metabolizing enzymes. Phospholipase A2 is considered to be involved as the rate-limiting step in the production of lipid mediators of inflammatory responses and, as such, it has been a target for drug design. A series of lipid mimetics including lipopeptides, amides and alcohols of lipidic alpha-amino acids, have been tested by bulk and monolayer assay techniques. The findings suggested the direct interaction of the tested compounds with porcine pancreatic phospholipase A2. The inactivation of the enzyme occurred in a competitive manner. The most active compound I (2-amino-N-hexadecyl-L-hexanamide) showed an apparent IC50 of 12 microM and inhibitory power Z = 13 in the monolayer assay.

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Competitive inhibition of lipolytic enzymes. I. A kinetic model applicable to water-insoluble competitive inhibitors

Cited by 86 publications

References 17 publications

Arginine 53 is involved in head‐group specificity of the active site of porcine pancreatic phospholipase A₂

Arginine 53 is involved in head‐group specificity of the active site of porcine pancreatic phospholipase A₂

Inactivation of pancreatic and gastric lipases by tetrahydrolipstatin and alkyl‐dithio‐5‐(2‐nitrobenzoic acid)

Synthesis and properties of novel lipopeptides and lipid mimetics

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Competitive inhibition of lipolytic enzymes. I. A kinetic model applicable to water-insoluble competitive inhibitors

Cited by 86 publications

References 17 publications

Arginine 53 is involved in head‐group specificity of the active site of porcine pancreatic phospholipase A2

Arginine 53 is involved in head‐group specificity of the active site of porcine pancreatic phospholipase A2

Inactivation of pancreatic and gastric lipases by tetrahydrolipstatin and alkyl‐dithio‐5‐(2‐nitrobenzoic acid)

Synthesis and properties of novel lipopeptides and lipid mimetics

Contact Info

Product

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Arginine 53 is involved in head‐group specificity of the active site of porcine pancreatic phospholipase A₂

Arginine 53 is involved in head‐group specificity of the active site of porcine pancreatic phospholipase A₂