Chemical modification of the histidine residue in phospholipase A2 (Naja naja naja). A case of half-site reactivity.

Roberts, Mary F.; Deems, Raymond A.; Mincey, Terry; Dennis, Edward A.

doi:10.1016/s0021-9258(17)40568-0

Cited by 202 publications

(28 citation statements)

References 21 publications

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“…Effect of Substrates and Activators on the Inactivation of Phospholipase A2 by p-Bromophenacyl Bromide. Phospholipase A2 can be inactivated by p-bromophenacyl bromide through the modification of 0.5 histidine per enzyme molecule (Roberts et al, 1977c). Triton with Ba2+ apparently inhibits the inactivation reaction by a combination of sequestering the reagent in the Triton micelles and altering enzyme conformation by metal ion binding.…”

Section: Resultsmentioning

confidence: 99%

“…Phosphatidylcholine activation is observed to lower the pXa controlling phospholipase A2 hydrolysis of phosphatidylethanolamine. A variety of phospholipids were examined for their ability to protect phospholipase A2 from p-bromophenacyl bromide inactivation (Roberts et al, 1977c). Interpretation of these data suggests the possibility that two functionally distinct phospholipid binding sites or subsites exist: an activator site which is specific for the lipid polar headgroup and a catalytic site which is nonspecific.…”

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confidence: 99%

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

Adamich¹,

Roberts²,

Dennis³

1979

Biochemistry

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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

Adamich¹,

Roberts²,

Dennis³

1979

Biochemistry

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“…Cobra venom PLA2 (Naja naja naja) was purified as described elsewhere (Hazlett & Dennis, 1985b;Reynolds & Dennis, 1991) and stored frozen at -20 °C. Inactivation of phospholipase A2 with p-bromophenacyl bromide (BPB) (Roberts et al, 1977) was previously described (Plesniak et al, 1993). All NMR solvents and deuterated dodecylphosphocholine (DODPC) were purchased from Cambridge Isotope Laboratories.…”

Section: Methodsmentioning

confidence: 99%

Conformation of micellar phospholipid bound to the active site of phospholipase A2

Plesniak

Lin²,

Dennis³

1995

Biochemistry

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Transferred NOE techniques have been used to determine the structure of phospholipid analogues bound to the active site of cobra venom phospholipase A2 (PLA2). These experiments were carried out on PLA2 with a substrate analogue which serves as an inhibitor, 1-(hexylthio)-2-(nonanoylamino)-1,2-dideoxy-sn-glycero-3-pho sphocholine (PC9). Because this inhibitor binds tightly to the enzyme and forms micelles at millimolar concentrations, experiments could be carried out to determine the conformation of the inhibitor when bound to the enzyme at the lipid-water interface. NOEs of the micellar lipid develop inefficiently in the absence of enzyme. NOESY experiments in the presence of PLA2 were used to determine the inhibitor structure and conformation when bound to the enzyme. The inhibitor adopts an active site conformation in which the end of the sn-2 chain is within 5 A of the alpha-methylene protons of the sn-1 chain. However, NOE cross-peaks in the experiments indicate that the backbone conformation of the bound lipid is different from that of a shorter chain lipid which forms monomers [Plesniak et al. (1993) Biochemistry 32, 5009-5016].

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“…456 Subsequent studies demonstrated its capability for labeling His residues. 536,537 Although still being used today, p-bromophenacyl bromide has been almost exclusively used in labeling phospholipase A 2 . 278,538,539 The most extensively used His labeling reagent to date is diethylpyrocarbonate (DEPC), whose reaction pathway with His is described in Scheme 7.…”

Section: Histidinementioning

confidence: 99%

“…p -Bromophenacyl bromide was first reported to react with carboxyl groups in Asp and Glu at the active sites of pepsin . Subsequent studies demonstrated its capability for labeling His residues. , Although still being used today, p -bromophenacyl bromide has been almost exclusively used in labeling phospholipase A 2 . ,, …”

Section: Targeted-labeling Reagentsmentioning

confidence: 99%

Mass Spectrometry-Based Protein Footprinting for Higher-Order Structure Analysis: Fundamentals and Applications

2020

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Proteins adopt different higher-order structures (HOS) to enable their unique biological functions. Understanding the complexities of protein higher-order structures and dynamics requires integrated approaches, where mass spectrometry (MS) is now positioned to play a key role. One of those approaches is protein footprinting. Although the initial demonstration of footprinting was for the HOS determination of protein/nucleic acid binding, the concept was later adapted to MS-based protein HOS analysis, through which different covalent labeling approaches “mark” the solvent accessible surface area (SASA) of proteins to reflect protein HOS. Hydrogen–deuterium exchange (HDX), where deuterium in D2O replaces hydrogen of the backbone amides, is the most common example of footprinting. Its advantage is that the footprint reflects SASA and hydrogen bonding, whereas one drawback is the labeling is reversible. Another example of footprinting is slow irreversible labeling of functional groups on amino acid side chains by targeted reagents with high specificity, probing structural changes at selected sites. A third footprinting approach is by reactions with fast, irreversible labeling species that are highly reactive and footprint broadly several amino acid residue side chains on the time scale of submilliseconds. All of these covalent labeling approaches combine to constitute a problem-solving toolbox that enables mass spectrometry as a valuable tool for HOS elucidation. As there has been a growing need for MS-based protein footprinting in both academia and industry owing to its high throughput capability, prompt availability, and high spatial resolution, we present a summary of the history, descriptions, principles, mechanisms, and applications of these covalent labeling approaches. Moreover, their applications are highlighted according to the biological questions they can answer. This review is intended as a tutorial for MS-based protein HOS elucidation and as a reference for investigators seeking a MS-based tool to address structural questions in protein science.

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Chemical modification of the histidine residue in phospholipase A2 (Naja naja naja). A case of half-site reactivity.

Cited by 202 publications

References 21 publications

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

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

Conformation of micellar phospholipid bound to the active site of phospholipase A2

Mass Spectrometry-Based Protein Footprinting for Higher-Order Structure Analysis: Fundamentals and Applications

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