Crystal structure of bovine pancreatic phospholipase A2 covalently inhibited by p-bromo-phenacyl-bromide

Renetseder, Roland; Dijkstra, Bauke W.; Huizinga, Kees; Kalk, Kor H.; Drenth, Jan

doi:10.1016/0022-2836(88)90342-7

Cited by 78 publications

(40 citation statements)

References 30 publications

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“…Since Tyr52 is fixed in an a-helix, this implies a displacement of residue Tyr69 towards the active center. The displacement of Tyr69 is in agreement with crystallographic studies on the p-bromophenacylbromide-inhibited enzyme [30]. In the monomeric crystallized complex the hydroxyl group of this tyrosine residue forms a hydrogen bond with the phosphate-group of the inhibitor.…”

Section: Discussionsupporting

confidence: 72%

Two‐dimensional ¹H‐NMR studies of phospholipase‐A₂‐inhibitor complexes bound to a micellar lipid‐water interface

Dekker

Peters

Slotboom

et al. 1991

European Journal of Biochemistry

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One-and two-dimensional NMR studies were performed on the complexes of porcine pancreatic phospholipase A2 with substrate analogs bound to a micellar lipid-water interface of fully deuterated dodecylphosphocholine. The interactions between the inhibitor and the enzyme were localized by comparison of the two-dimensional NOE spectra recorded for the enzyme-inhibitor complex using both protonated and selectively deuterated inhibitors. These experiments led us to the following conclusions for the phospholipase-A,-micelle complex: (i) the 38-kDa phospholipase A2 complex gives NMR spectra with relatively narrow lines, which is indicative of high mobility of the enzyme; (ii) the residues Alal, Trp3, Phe63 and Tyr69 located in the interface recognition site, as well as Phe22, Tyr75, Phel06 and Tyrl 1 1 are involved in the micelle-binding process; (iii) when present on the micelle, phospholipase A2 is stereospecific for the inhibitor binding; (iv) the inhibitor, (R)-dodecyl-2-aminohexanol-lphosphoglycol, binds stoichiometrically to phospholipase A2 with high affinity (Kd 5 10 pM); (v) the inhibitor binds in the active site of the enzyme, which is evidenced by large chemical-shift differences for Phe5, Ile9, Phe22, His48, Tyr52 and Phel06; (vi) the acyl chain of the inhibitor makes hydrophobic contacts ( < 0.4 nm) near Phe5, Ile9, Phe22 and Phel06.Comparison of our results on the enzyme-inhibitor-micelle ternary complex with the crystal structure of the enzyme-inhibitor complex [Thunnissen, M.

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Section: Discussionsupporting

confidence: 72%

Two‐dimensional ¹H‐NMR studies of phospholipase‐A₂‐inhibitor complexes bound to a micellar lipid‐water interface

Dekker

Peters

Slotboom

et al. 1991

European Journal of Biochemistry

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“…However, these arguments cannot be accepted as definite because modified binding properties could change the interpretation. Indeed alkylation by p-bromophenacylbromide is known to potentially modify the structure of the enzymes as shown for instance for the pancreatic enzyme (Renetseder et al, 1988) or a toxic sPLA 2 , notexin (Pluskal et al, 1978). On the other hand, AML was found to significantly compete with ATX A only on Torpedo synaptosomes (Pungercar et al, 1998) and weakly on mammalian brain preparations (Krizaj et al, 1995).…”

Section: Discussionmentioning

confidence: 97%

Secreted phospholipase A2-induced neurotoxicity and epileptic seizures after intracerebral administration: An unexplained heterogeneity as emphasized with paradoxin and crotoxin

et al. 1998

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After intracerebral injection, some toxic secreted phospholipases A2 (sPLA2) can induce epileptic seizures which bases are currently ill known. We undertook the detailed study of the central neurotoxicity of paradoxin (PDX), an analog of taipoxin, in rodents. Since literature strongly suggests a high variability in the sPLA2 epileptogenic properties, we compared, in an acute model, PDX with crotoxin (CTX), known to induce seizures and that may bind to similar neuronal receptors. Related toxic enzymes (ammodytoxin A, ATX A, and CTX subunit CB) and the non neurotoxic sPLA2 from pancreas and PLA2 analog ammodytin L (AML) were also tested. Despite being highly neurotoxic, PDX did not induce either convulsions or long-lasting seizure fits. The results obtained with the other enzymes showed that toxic sPLA2s can effectively be differentiated based on two criteria: the presence of cortically recorded epileptic paroxysmal discharges (E) and convulsions (C). We thus propose to classify the toxic sPLA2s into different groups depending on their epileptogenic properties: E-C-(PDX), E+C+ (CTX, CB), and E-C+ (ATX A). The non toxic AML and pancreatic enzyme were E-C-. Moreover, the results obtained with AML, and preliminarily with chemically inhibited CB, suggested that phospholipid hydrolysis is important to trigger seizures and convulsions. However, PDX and CTX that possess highly different epileptogenic properties exerted comparable, although slightly different, catalytic activities. Similarly, histological evaluations of the brain of PDX and CTX-treated rats (H&E staining, GFAP immunodetection, hsp70 and c-fos mRNA detection) did not provide satisfactory clues to explain these large differences. Further studies are strongly required.

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“…It could be envisaged that the side chain at position 53 in the native enzyme and the mutants is sufficiently flexible to allow for a large variation in orientation. It has been shown, for example, that the side chain of Tyr69, which points into the active site in the enzyme-inhibitor complex of porcine pancreatic PLA, (Thunnissen et al, 1990), can adopt an outward confo,rmation in the apo-enzyme, resulting in considerable movement of its phenolic hydroxyl group (Renetseder et al, 1988). It is, however, difficult to explain how an outward orientation of, for example Glu53 would improve the interaction with negatively charged substrates.…”

Section: Discussionmentioning

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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Crystal structure of bovine pancreatic phospholipase A2 covalently inhibited by p-bromo-phenacyl-bromide

Abstract: Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.

Cited by 78 publications

References 30 publications

Two‐dimensional ¹H‐NMR studies of phospholipase‐A₂‐inhibitor complexes bound to a micellar lipid‐water interface

Two‐dimensional ¹H‐NMR studies of phospholipase‐A₂‐inhibitor complexes bound to a micellar lipid‐water interface

Secreted phospholipase A2-induced neurotoxicity and epileptic seizures after intracerebral administration: An unexplained heterogeneity as emphasized with paradoxin and crotoxin

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

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Crystal structure of bovine pancreatic phospholipase A2 covalently inhibited by p-bromo-phenacyl-bromide

Abstract: Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.

Cited by 78 publications

References 30 publications

Two‐dimensional 1H‐NMR studies of phospholipase‐A2‐inhibitor complexes bound to a micellar lipid‐water interface

Two‐dimensional 1H‐NMR studies of phospholipase‐A2‐inhibitor complexes bound to a micellar lipid‐water interface

Secreted phospholipase A2-induced neurotoxicity and epileptic seizures after intracerebral administration: An unexplained heterogeneity as emphasized with paradoxin and crotoxin

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

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Two‐dimensional ¹H‐NMR studies of phospholipase‐A₂‐inhibitor complexes bound to a micellar lipid‐water interface

Two‐dimensional ¹H‐NMR studies of phospholipase‐A₂‐inhibitor complexes bound to a micellar lipid‐water interface

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