Effects of substitution of the absolutely invariant Gln-4 and Phe-5 in bovine pancreatic phospholipase A2 on enzymic activity and substrate binding properties

Gj, van Scharrenburg; Wc, Puijk; Mr, Egmond; Schaft, van der Arjan; Gh, de Haas; Aj, Slotboom

doi:10.1021/bi00535a037

Cited by 25 publications

(14 citation statements)

References 44 publications

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“…The egg yolk assay was carried out according to the modified procedure described in [24]. Kinetic measurements using micellar 1,2-dioctanoyl-sn-glycero-3-phosphocholine and monomeric rat-2,3-bis(hexanoylthio)-propyl-phosphocholine were performed as described [20,25].…”

Section: Kinetic Measurementsmentioning

confidence: 99%

Synthetic peptide from lipocortin I has no phospholipase A₂ inhibitory activity

Binsbergen¹,

Slotboom²,

Aarsman³

1989

FEBS Letters

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Two anti-inflammatory peptides corresponding to a high amino acid similarity region between lipocortins were synthesized and tested on their ability to inhibit porcine pancreatic phospholipase &. Kinetic assays using monomeric and aggregated phospholipids did not reveal any phospholipase A, inhibitory activity. The peptides did not inhibit phospholipase A2 activity on monolayers of negatively charged substrate and did not prevent phospholipase 4 action on mixed micelles of l-stearoyl-2-arachidonoyl-sn-glycero-3-phosph~holine and sodiumdeoxycholate. Ultraviolet difference spectroscopy did not show binding of the peptides to phospholipase 4. Therefore we conclude that these anti-inflammatory peptides do not inhibit pancreatic phospholipase A2 in vitro, in contrast to the results recently published [(1988) Nature 335, 7267301.

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Section: Kinetic Measurementsmentioning

confidence: 99%

Synthetic peptide from lipocortin I has no phospholipase A₂ inhibitory activity

Binsbergen¹,

Slotboom²,

Aarsman³

1989

FEBS Letters

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“…Trp-3 is conserved among pancreatic PLA2s, but it is usually a different hydrophobic residue in other types of PLA2. The spectroscopic properties of Trp-3 have been used to show that the N-terminus is indeed perturbed upon interfacial binding (van Scharrenburg et al, 1982(van Scharrenburg et al, , 1984aRamirez & Jain, 1991). Position 6 is usually an arginine in pancreatic PLA2 from other animals (Davidson & Dennis, 1990;Heinrikson, 1991).…”

mentioning

confidence: 99%

Phospholipase A2 engineering. Probing the structural and functional roles of N-terminal residues with site-directed mutagenesis, x-ray, and NMR

Liu

Zhu²,

Huang³

et al. 1995

Biochemistry

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The N-terminal residues of phospholipase A2 (PLA2) are believed to be involved in the hydrogen-bonding network, the interfacial binding site, or the hydrophobic channel. Site-directed mutants of bovine pancreatic PLA2 with substitutions at positions 2, 3, 4, 5, 6, and 9 were constructed to test the roles of these residues in the structure and function of PLA2. Nonconservative mutations of Phe-5 and Ile-9, which are located inside the hydrophobic channel, led to significant perturbations in the conformation and conformational stability. Kinetic studies also indicated that mutations at Ile-9 and Phe-5 caused significant decreases in the rate of hydrolysis toward micellar and vesicle substrates. Scooting mode kinetic analysis showed that the binding step of the mutant enzymes to the DC14PM (1,2-dimyristoyl-sn-glycero-3-phosphomethanol) vesicle interface is not significantly affected and that the perturbations in catalysis occur mainly in kcat at the interface. The results taken together suggest that the residues Ile-9 and Phe-5 are important for both structure and catalysis. The mutant W3A (Trp-3 to Ala) also showed decreased rates of hydrolysis but to a lesser extent than Ile-9 and Phe-5 mutants. In addition, the binding affinity of W3A to the surface of the vesicles (i.e., the E to E* step) has been perturbed to the extent that hopping between anionic vesicles has been observed. On the other hand, the mutants of Gln-4 and Asn-6, which are located at or near the surface, displayed structural and kinetic properties similar to those of the wild-type PLA2 with the exception of the highly hydrophilic lysine mutant. The X-ray structure of the Q4E mutant indicates that the overall structure, the catalytic triad, and the link between residue 4 and Asp-99 via hydrogen bonding through Ala-1 and the structural water remain the same as in the WT. Substitutions for Leu at position 2 showed an acyl chain length discrimination toward different substrates, which may reflect the contacting position(s) of the substrate acyl chain with Leu-2.

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“…As fosfolipases A 2 (FLA 2 ) constituem uma família de proteínas estruturalmente relacionadas, capazes de hidrolisar a ligação acíl-éster na posição sn-2 de fosfoglicerídeos e liberar quantidades equimolares de ácidos graxos livres, como ácido araquidônico e lisofosfolipídeos (van Scharrenburg et al, 1982;Dennis, 1994). O ácido aracdônico é substrato para biossíntese de mediadores lipídicos envolvidos na resposta inflamatória, tais como prostaglandinas, leucotrienos e fatores de agregação plaquetária (Parente et al, 2001) que são capazes de afetar a permeabilidade vascular e o fluxo de sangue da microcirculação promovendo a infiltração celular para o sítio inflamatório, além de promover dano tecidual pela liberação de enzimas lisossomais, proteínas catiônicas e espécies reativas de oxigênio (artigo para revisão) (Harris, 1993).…”

Section: Lista De Abreviaturas E Siglasunclassified

Caracterização da inflamação articular induzida por fosfolipase A2 - grupo II A: determinação das alterações histopatológicas, comportamentais e mediação química.

Dias¹

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Effects of substitution of the absolutely invariant Gln-4 and Phe-5 in bovine pancreatic phospholipase A2 on enzymic activity and substrate binding properties

Cited by 25 publications

References 44 publications

Synthetic peptide from lipocortin I has no phospholipase A₂ inhibitory activity

Synthetic peptide from lipocortin I has no phospholipase A₂ inhibitory activity

Phospholipase A2 engineering. Probing the structural and functional roles of N-terminal residues with site-directed mutagenesis, x-ray, and NMR

Caracterização da inflamação articular induzida por fosfolipase A2 - grupo II A: determinação das alterações histopatológicas, comportamentais e mediação química.

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Effects of substitution of the absolutely invariant Gln-4 and Phe-5 in bovine pancreatic phospholipase A2 on enzymic activity and substrate binding properties

Cited by 25 publications

References 44 publications

Synthetic peptide from lipocortin I has no phospholipase A2 inhibitory activity

Synthetic peptide from lipocortin I has no phospholipase A2 inhibitory activity

Phospholipase A2 engineering. Probing the structural and functional roles of N-terminal residues with site-directed mutagenesis, x-ray, and NMR

Caracterização da inflamação articular induzida por fosfolipase A2 - grupo II A: determinação das alterações histopatológicas, comportamentais e mediação química.

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Synthetic peptide from lipocortin I has no phospholipase A₂ inhibitory activity

Synthetic peptide from lipocortin I has no phospholipase A₂ inhibitory activity