Phospholipid activation of cobra venom phospholipase A2. 2. Characterization of the phospholipid-enzyme interaction

Adamich, Marina; Roberts, Mary F.; Dennis, Edward A.

doi:10.1021/bi00582a017

Cited by 66 publications

(34 citation statements)

References 25 publications

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“…However, the experimental data obtained between pH 5.5 and 9.0 all fit best to a Hill equation with the Hill coefficient of 1.66, indicating that substrate binds to the enzyme in a cooperative manner at the interface. Previous studies have suggested that the enzyme has two binding sites (a catalytic and an activator site) and phosphatidylcholine substrates bind to both (18)(19)(20). Whether an additional binding step should be considered for the hydrolysis of thio-PC is not clear at this time.…”

Section: Resultsmentioning

confidence: 95%

Critical role of a hydrogen bond in the interaction of phospholipase A2 with transition-state and substrate analogues.

Lin

Dennis

1991

Proc. Natl. Acad. Sci. U.S.A.

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The inhibition of phospholipase A2 by an amide substrate analogue, 1-hexadecylthio-2-hexadecanoylamino-1,2-dideoxy-sn-glycero-3-phosphocholine, and a phosphonate transition-state analogue, 1-hexadecylthio-1-deoxy-2-hexadecylphosphono-sn-glycero-3-phosphocholine, is dramatically influenced by pH. However, these two inhibitors show opposite pH dependencies. The amide analogue acts more potently under basic conditions, whereas the phosphonate acts more potently under acidic conditions. In both cases, lgand binding is perturbed by protonation of an enzyme functional group with an apparent pK. of 6.1, which corresponds to that of a histidine residue. Thus, His-48, which has previously been implicated in catalysis, appears to be critically involved in the hydrogen bond interactions between the enzyme and these two inhibitors. The amide analogue binds most effectively to the enzyme when His-48 is deprotonated. Upon protonation of the histidine residue, the amide cannot form a critical hydrogen bond and loses its ability to interact effectively with the enzyme.In contrast, the phosphonate analogue binds much tighter to the protonated form of the enzyme than to the deprotonated form. The phosphonate analogue needs a bridging hydrogen between the oxygen on its phosphorus atom and the N81 of His-48 to form a strong hydrogen bond. At optimal pH values for inhibitor binding, both the amide and the phosphonate analogues are potent competitive inhibitors of cobra (Naja naja naja) venom phospholipase A2. The IC5o for the amide was 4.4 x 10-4 mol fraction and for the phosphonate was 1.6 x 10-5 mol fraction. Under the experimental conditions used, this corresponds to a bulk concentration of 2 IpM and 70 nM, respectively.Recently, two types of reversible, tight-binding phospholipase A2 inhibitors have been rationally designed and characterized. The first type is composed of amide analogues in which an amide functional group is used to replace the scissile ester bond at the sn-2 position of phospholipids (1, 2). The other is composed of phosphonate analogues in which a tetrahedral phosphonate group is used in place of the sn-2 ester bond of phospholipids to mimic the putative transition state of phospholipase A2-catalyzed hydrolysis (3). Crystallographic studies of phospholipase A2 complexed with these analogues have suggested that both inhibitors interact with the enzyme via a strong hydrogen bond (4-6). These studies indicate that the amide analogue forms a hydrogen bond through the amide hydrogen with the N61 atom of His-48.The phosphonate analogue forms a hydrogen bond through one of the nonbridging oxygen atoms of the phosphonate group with the protonated N81 atom of His-48. In this manuscript, we explore the ramifications of these findings on the binding of various inhibitors to the enzyme.The concept of transition-state analogues has been successfully applied to develop potent inhibitors of a variety of enzymes (7). Such transition-state analogues have been designed based on the premise that an enzyme catalyzes a rea...

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Section: Resultsmentioning

confidence: 95%

Critical role of a hydrogen bond in the interaction of phospholipase A2 with transition-state and substrate analogues.

Lin

Dennis

1991

Proc. Natl. Acad. Sci. U.S.A.

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“…This enzyme has also been shown to be activated by phospholipids containing phosphatidylcholine (PC) head groups [56], and two possible sites for this interaction have been suggested [41, 57]. A combination of site-directed mutagenesis and equilibrium dialysis has identified and confirmed there is an activator site distinct from the catalytic site [58, 59].…”

Section: Group Ia Pla2mentioning

confidence: 99%

“…The majority of sPLA 2 enzymes preferentially hydrolyze anionic substrates [18]. The GIA enzyme however is able to hydrolyze zwitterionic substrate equally as well as negatively charged lipid surfaces [56, 61]. This is most likely due to the aromatic residues present on the interfacial binding surface of the GIA PLA 2 as shown in Fig.…”

Section: Group Ia Pla2mentioning

confidence: 99%

Phospholipase A2 Biochemistry

Burke

Dennis

2008

Cardiovasc Drugs Ther

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358

276

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The phospholipase A 2 (PLA 2 ) superfamily consists of many different groups of enzymes that catalyze the hydrolysis of the sn-2 ester bond in a variety of different phospholipids. The products of this reaction, a free fatty acid, and lysophospholipid have many different important physiological roles. There are five main types of PLA 2 : the secreted sPLA 2 's, the cytosolic cPLA 2 's, the Ca 2+ independent iPLA 2 's, the PAF acetylhydrolases, and the lysosomal PLA 2 's. This review focuses on the superfamily of PLA 2 enzymes, and then uses three specific examples of these enzymes to examine the differing biochemistry of the three main types of these enzymes. These three examples are the GIA cobra venom PLA 2 , the GIVA cytosolic cPLA 2 , and the GVIA Ca 2+ -independent iPLA 2 .

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“…membrane. It has been reported (Adamich et al, 1979) that the preference of N. naja phospholipase A2 for phosphatidylcholine over phosphatidylethanolamine can be reversed by presenting both components together in an optimally heterogeneous mixture. To determine if the macrophage enzymes exhibit differential rates of hydrolysis similar to that observed in N. naja phospholipase A2, the hydrolysis of phosphatidylethanolamine, phosphatidylcholine or phosphatidylinositol alone or combined with the polar lipids normally present in a biological membrane was measured.…”

Section: Determination Ofrates Ofsubstrate Hydrolysismentioning

confidence: 99%

[Identification and characterization of two phospholipase A2 activities in resident mouse peritoneal macrophages.]

Wightman¹,

Humes²,

Davies³

et al. 1981

Biochemical Journal

115

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Resident mouse peritoneal macrophages synthesize and release large amounts of prostaglandins in response to inflammatory stimuli. Release of prostaglandin E2 and 6-oxoprostaglandin F1 alpha occurs at a rate of 1 nmol/h per mg of cell protein. The mechanisms by which substrate arachidonic acid is released have yet to be established. We have therefore initiated studies to characterize those enzymes that can catalyse its release from phospholipid and may be of significance at the cellular level. We report initially the characterization of two phospholipase A2 activities in homogenates of mouse peritoneal macrophages. The first is active at pH 4.5 and is not dependent on Ca2+. The second is Ca2+-dependent and is optimally active at pH 8.5. Either phospholipase A2 activity is capable of hydrolysing [14C] arachidonic acid from [14C] arachidonic acid-labelled phospholipids in quantities sufficient to account for the amounts of prostaglandins by macrophages in culture. Phospholipid substrates are prepared from mouse LM fibroblasts in serum-free Higuchi medium containing radiolabelled phospholipid precursors. Single-labelled phospholipids bear the 14C label in the arachidonic acid moiety. Dual-labelled phospholipids bear a 14C label in the polar head group and a 3H label in the arachidonic acid moiety. Experiments with dual-labelled substrates establish that both phospholipase activities are of the A2 type as indicated by the equimolar recovery of [3H] arachidonic acid and [14C] lysophospholipid. Studies with aqueous sonicated dispersions of purified [14C] arachidonic acid-labelled phospholipid or mixed liposomal substrates formed from mixtures of cellular polar lipids reveal that the pH 4.5 activity hydrolyses phosphatidylethanolamine and phosphatidylcholine more efficiently when they are present in a mixture of other polar lipids. The pH 8.5 activity, however, hydrolyses the purified phospholipids more efficiently.

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Phospholipid activation of cobra venom phospholipase A2. 2. Characterization of the phospholipid-enzyme interaction

Cited by 66 publications

References 25 publications

Critical role of a hydrogen bond in the interaction of phospholipase A2 with transition-state and substrate analogues.

Critical role of a hydrogen bond in the interaction of phospholipase A2 with transition-state and substrate analogues.

Phospholipase A2 Biochemistry

[Identification and characterization of two phospholipase A2 activities in resident mouse peritoneal macrophages.]

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