Lipidomics Reveals Dramatic Physiological Kinetic Isotope Effects during the Enzymatic Oxygenation of Polyunsaturated Fatty Acids Ex Vivo

Arachidonic acid (AA, 20:4) is an omega-6 polyunsaturated fatty acid (PUFA) and the main precursor to the class of lipid mediators known as eicosanoids. The enzymes that catalyze the oxygenation of AA begin by abstracting hydrogen from one of three bis-allylic carbons within 1,4-<i>cis</i>,<i>cis</i>-diene units. Substitution of deuterium for hydrogen has been shown to lead to massive kinetic isotope effects (KIE) for soybean lipoxygenase (sLOX) oxygenation of linoleic acid (LA, 18:2). Yet, experimental determination of the KIE during oxygenation of AA and LA by mammalian enzymes including cyclooxygenase (COX) and lipoxygenase (LOX) has revealed far lower values. All prior studies investigating the KIE of PUFA oxygenation have relied on <i>in vitro</i> systems using purified enzymes and were limited by availability of deuterated substrates. Here we demonstrate the use of macrophages as an <i>ex vivo</i> model system to study the physiological KIE (PKIE) during enzymatic AA oxygenation by living cells using a newly synthesized library of deuterated AA isotopologues. By extending lipidomic UPLC-MS/MS approaches to simultaneously quantify native and deuterated lipid products, we were able to demonstrate that the magnitude of the PKIE measured in macrophages for COX and LOX oxygenation of AA is similar to KIEs determined in previous reports using the AA isotopologue deuterated at carbon 13 (C13). However, for the first time we show that increasing the number of deuterated bis-allylic carbons to include both C10 and C13 leads to a massive increase in the PKIE for COX oxygenation of AA. We provide evidence that hydrogen(s) present at C10 of AA play a critical role in the catalysis of prostaglandin and thromboxane synthesis. Furthermore, we discovered that deuteration of C10 promotes the formation of the resolving lipid mediator lipoxin B4, likely by interfering with AA cyclization and shunting AA to the LOX pathway under physiological conditions

Cardiolipin Effects on PLA₂ Activity toward PAPC.

<p>The <i>in vitro</i> mixed micelle assay was utilized to determine if cardiolipin affects the enzymatic activities of A. GVIA iPLA₂, B. GIA sPLA₂, C. GV sPLA₂, and D. GIVA cPLA₂ toward PAPC. Mixed micelles composed of 100 µM phospholipid and 400 µM Triton X-100 was employed as substrate containing the mole % of cardiolipin to PAPC indicated.</p

Membrane Allostery and Unique Hydrophobic Sites Promote Enzyme Substrate Specificity

We demonstrate that lipidomics coupled with molecular dynamics reveal unique phospholipase A₂ specificity toward membrane phospholipid substrates. We discovered unexpected headgroup and acyl-chain specificity for three major human phospholipases A₂. The differences between each enzyme’s specificity, coupled with molecular dynamics-based structural and binding studies, revealed unique binding sites and interfacial surface binding moieties for each enzyme that explain the observed specificity at a hitherto inaccessible structural level. Surprisingly, we discovered that a unique hydrophobic binding site for the cleaved fatty acid dominates each enzyme’s specificity rather than its catalytic residues and polar headgroup binding site. Molecular dynamics simulations revealed the optimal phospholipid binding mode leading to a detailed understanding of the preference of cytosolic phospholipase A₂ for cleavage of proinflammatory arachidonic acid, calcium-independent phospholipase A₂, which is involved in membrane remodeling for cleavage of linoleic acid and for antibacterial secreted phospholipase A₂ favoring linoleic acid, saturated fatty acids, and phosphatidylglycerol

Monitoring with strong interests and weak incentives in Palawan, the Philippines

We demonstrate that lipidomics coupled with molecular dynamics reveal unique phospholipase A₂ specificity toward membrane phospholipid substrates. We discovered unexpected headgroup and acyl-chain specificity for three major human phospholipases A₂. The differences between each enzyme’s specificity, coupled with molecular dynamics-based structural and binding studies, revealed unique binding sites and interfacial surface binding moieties for each enzyme that explain the observed specificity at a hitherto inaccessible structural level. Surprisingly, we discovered that a unique hydrophobic binding site for the cleaved fatty acid dominates each enzyme’s specificity rather than its catalytic residues and polar headgroup binding site. Molecular dynamics simulations revealed the optimal phospholipid binding mode leading to a detailed understanding of the preference of cytosolic phospholipase A₂ for cleavage of proinflammatory arachidonic acid, calcium-independent phospholipase A₂, which is involved in membrane remodeling for cleavage of linoleic acid and for antibacterial secreted phospholipase A₂ favoring linoleic acid, saturated fatty acids, and phosphatidylglycerol

Differential Cardiolipin Hydrolysis by iPLA₂ and sPLA₂.

<p>The hydrolysis of cardiolipin by A. GVIA iPLA₂, B. GIA sPLA₂ and C. GV sPLA₂ were examined in mixed micelle assays containing 100 µM cardiolipin and 400 µM Triton X-100 over a 100 min time course. The cardiolipin (green), monolyso- (red) and dilyso-cardiolipin (blue) were measured in the same samples by LC/MS. The percentages of cardiolipin and lyso-cardiolipin are based on ion intensity counts.</p

Structure of Cardiolipin.

<p>The structure of cardiolipin, 1′,3′-Bis-[1, 2-di-(9Z-octadecenoyl)-sn-glycero-3-phopho]-<i>sn</i>-glycerol, is drawn and adapted from LIPID MAPS (<a href="http://www.lipidmaps.org" target="_blank">www.lipidmaps.org</a>).</p

Membrane Allostery and Unique Hydrophobic Sites Promote Enzyme Substrate Specificity

We demonstrate that lipidomics coupled with molecular dynamics reveal unique phospholipase A₂ specificity toward membrane phospholipid substrates. We discovered unexpected headgroup and acyl-chain specificity for three major human phospholipases A₂. The differences between each enzyme’s specificity, coupled with molecular dynamics-based structural and binding studies, revealed unique binding sites and interfacial surface binding moieties for each enzyme that explain the observed specificity at a hitherto inaccessible structural level. Surprisingly, we discovered that a unique hydrophobic binding site for the cleaved fatty acid dominates each enzyme’s specificity rather than its catalytic residues and polar headgroup binding site. Molecular dynamics simulations revealed the optimal phospholipid binding mode leading to a detailed understanding of the preference of cytosolic phospholipase A₂ for cleavage of proinflammatory arachidonic acid, calcium-independent phospholipase A₂, which is involved in membrane remodeling for cleavage of linoleic acid and for antibacterial secreted phospholipase A₂ favoring linoleic acid, saturated fatty acids, and phosphatidylglycerol

Membrane Allostery and Unique Hydrophobic Sites Promote Enzyme Substrate Specificity

We demonstrate that lipidomics coupled with molecular dynamics reveal unique phospholipase A₂ specificity toward membrane phospholipid substrates. We discovered unexpected headgroup and acyl-chain specificity for three major human phospholipases A₂. The differences between each enzyme’s specificity, coupled with molecular dynamics-based structural and binding studies, revealed unique binding sites and interfacial surface binding moieties for each enzyme that explain the observed specificity at a hitherto inaccessible structural level. Surprisingly, we discovered that a unique hydrophobic binding site for the cleaved fatty acid dominates each enzyme’s specificity rather than its catalytic residues and polar headgroup binding site. Molecular dynamics simulations revealed the optimal phospholipid binding mode leading to a detailed understanding of the preference of cytosolic phospholipase A₂ for cleavage of proinflammatory arachidonic acid, calcium-independent phospholipase A₂, which is involved in membrane remodeling for cleavage of linoleic acid and for antibacterial secreted phospholipase A₂ favoring linoleic acid, saturated fatty acids, and phosphatidylglycerol

Membrane Allostery and Unique Hydrophobic Sites Promote Enzyme Substrate Specificity

We demonstrate that lipidomics coupled with molecular dynamics reveal unique phospholipase A₂ specificity toward membrane phospholipid substrates. We discovered unexpected headgroup and acyl-chain specificity for three major human phospholipases A₂. The differences between each enzyme’s specificity, coupled with molecular dynamics-based structural and binding studies, revealed unique binding sites and interfacial surface binding moieties for each enzyme that explain the observed specificity at a hitherto inaccessible structural level. Surprisingly, we discovered that a unique hydrophobic binding site for the cleaved fatty acid dominates each enzyme’s specificity rather than its catalytic residues and polar headgroup binding site. Molecular dynamics simulations revealed the optimal phospholipid binding mode leading to a detailed understanding of the preference of cytosolic phospholipase A₂ for cleavage of proinflammatory arachidonic acid, calcium-independent phospholipase A₂, which is involved in membrane remodeling for cleavage of linoleic acid and for antibacterial secreted phospholipase A₂ favoring linoleic acid, saturated fatty acids, and phosphatidylglycerol

The phospholipase A₂ superfamily.

<p>Adapted from Dennis et al <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059267#pone.0059267-Dennis1" target="_blank">[1]</a>.</p