Interfacial Catalysis: the Mechanism of Phospholipase A
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Scott, David; White, Steven P.; Otwinowski, Zbyszek; Wei, Yuan; Gelb, Michael H.; Sigler, Paul B.

doi:10.1126/science.2274785

Cited by 741 publications

(588 citation statements)

References 45 publications

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“…These residues form the 'lipid binding domain' which is located at one side of the protein (for a review see Waite, 1987). X-ray studies on monomer lipid binding in the active site of PLA, from porcine pancreas (Thunnissen et al, 1990), cobra and bee venom (Scott et al, 1990) and human secretory PLA, (Scott et al, 1991) have been performed. Thunnissen et al (1990) showed for (mutant) porcine pancreatic PLA, contacts between 91-69 and the phosphate group of the inhibitor and a possible contact between the hydroxyl group of the glycol and Asp49.…”

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confidence: 99%

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 Arg53 has been replaced by a glutamine and a glutamic acid in mutants R53Q and R53E, respectively.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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mentioning

confidence: 99%

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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“…All catalytically active sPLA # s are known to have a Ca# + -binding loop with a highly conserved consensus motif, Xaa-Cys-Gly-Xaa-Gly [26]. Gly$!…”

Section: Ef Does Not Bind Heparin Via C-terminal Cationic Residuesmentioning

confidence: 99%

Enhancing activity and phospholipase A2 activity: two independent activities present in the enhancing factor molecule

Kadam

Mulherkar

1999

Biochemical Journal

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Enhancing factor (EF), a molecule that increases the binding of epidermal growth factor (EGF) to A431 cells, was first isolated in our laboratory from mouse intestines, and subsequently shown to be a secretory form of phospholipase A2 (PLA2) [Mulherkar, Rao, Wagle, Patki and Deo (1993) Biochem. Biophys. Res. Commun. 195, 1254-1263]. We had proposed earlier that EF increases the binding of EGF by first binding to its own cell-surface receptor [identified as a 100 kDa molecule; Mulherkar and Deo (1986) J. Cell. Physiol. 127, 183-188], and then by creating a binding site for EGF. However, due to its PLA2 activity, there was a possibility that EF, by its phospholipase activity could be unmasking cryptic EGF receptors on the cell surface, thereby increasing the number of binding sites for EGF. To test whether enhancing activity and phospholipase activity are independent of each other, a series of mutations were created using the full-length EF cDNA as a template, expressed in 293 cells and the mutant recombinant proteins checked for EF as well as PLA2 activities. Our studies have shown that one of the mutant EF proteins, lacking PLA2 activity, retains EF activity. This demonstrates unambiguously that EF and PLA2 activities are two independent activities in the same molecule. Mutation in the Ca2+-binding loop resulted in loss of EF activity, thereby demonstrating that EF activity is Ca2+-dependent. The N-terminal region of the EF molecule appears to be crucial for the enhancing activity.

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“…Entre la fosfolipasa A, de las abejas y la fosfolipasa A,B de las avispas existe poca similitud en sus secuencias de aa y no tienen alergenicidad cruzada (66). El sitio catalítico de Api m 1 es similar al de la fosfolipasa A, de la cobra china (Naja naja atra) (68,69). Sin embargo, poco se conoce sobre la capacidad alergénica de las fosfolipasas de este reptil.…”

Section: Fosfolipasasunclassified

Alergenos: relación entre función biológica y alergenicidad

1998

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ResumenLos alergenos se encuentran en muy diversas fuentes, pero, tienen la particularidad de inducir la producción de inmunoglobulina E (IgE) y provocar alergia. No se conoce una característica molecular biológica común entre los alergenos que explique su capacidad alergénica. En los últimos años, se han demostrado o inferido diferentes funciones biológicas en los alergenos, generando nuevas hipótesis sobre el papel de dichas funciones en la actividad alergénica. Con el fin de identificar qué funciones biológicas se han demostrado o inferido en los alergenos y analizar su posible influencia en el papel alergénico, se revisaron aproximadamente doscientos alergenos teniendo como base los ya caracterizados por el Comité de Nomenclatura de Alergenos de la OMS, como también otros menos caracterizados pero con algunas propiedades fisico~~liímicas conocidas. Los alergenos se agruparon de acuerdo con la actividad biológica y se tabularon con su fuente de origen, peso molecular (PM), punto isoeléctrico (pl), frecuencia de reactividad en la población alérgica, potenciales sitios de glicosilación y formación de enlaces disulfuro. Se identificaron 88 alergenos en los que se ha informado alguna actividad biológica. El agrupamiento de estos según la actividad produjo los siguientes resultados: enzimática (47,2%), inhibición de enzimas (11,3%), transporte (18,1%), regulación de la actividad celular (15,9%) y otras actividades como la de conferir resistencia a enfermedades en plantas y citólisis (7,9%). En muchos de estos alergenos, la actividad biológica ha sido inferida por su homología estructural con proteínas de función conocida, pero los experimentos que corroboren dicha función no se han realizado. La mayoría de estos alergenos tienen en común PM<60 kDa, p1<7,0 y escasa formación de oligómeros. Nuestro análisis sugiere que la capacidad de inducir alergia no está determinada por ninguna de las funciones biológicas descritas. Aunque la actividad de cisterna-proteasa y serina-proteasa de algunos de los alergenos de los ácaros domésticos han sido señaladas como determinantes en el papel alergénico en dichos alergenos, los datos experimentales no son concluyentes y no permiten atribuir la alergenicidad a la función enzimática. Allergens: the relationship between biological function and allergenicityAllergens can be found from many diverse sources but they have the particular quality of being able to induce immunoglobulin E (IgE) production and to provoke allergy. A common biological molecular characteristic which can explain their allergenic capacity is not known amongst the allergens. In the last few years allergens' different biological functions have been demonstrated or inferred, generating a new hypothesis about the role of the said functions in allergenic activity. To identify which biological functions have Instituto de Investigaciones Inmunológicas, Universidad de Cartagena Recibido para su publicación: enero 15 de 1998.

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Interfacial Catalysis: the Mechanism of Phospholipase A ₂

Cited by 741 publications

References 45 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

Enhancing activity and phospholipase A2 activity: two independent activities present in the enhancing factor molecule

Alergenos: relación entre función biológica y alergenicidad

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Interfacial Catalysis: the Mechanism of Phospholipase A 2

Cited by 741 publications

References 45 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

Enhancing activity and phospholipase A2 activity: two independent activities present in the enhancing factor molecule

Alergenos: relación entre función biológica y alergenicidad

Contact Info

Product

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Interfacial Catalysis: the Mechanism of Phospholipase A ₂

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