Defining the dimensions of the catalytic site of phospholipase A2 using amide substrate analogs

Lin, Yu; Dennis, Edward A.

doi:10.1021/ja00049a001

Cited by 39 publications

(38 citation statements)

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“…Furthermore, the information could also be biased, since studies with other lipid-binding proteins have shown that the mode of accommodation of an alkyl chain can be markedly influenced by the other alkyl chain of the bound lipid (14,15). Another uncertainty with non-cleavable lipid analogues is that the "unnatural" moieties may greatly increase their affinity for the enzyme (16), thus potentially masking effects of acyl chain structure.…”

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confidence: 99%

Substrate Efflux Propensity Plays a Key Role in the Specificity of Secretory A-type Phospholipases

Haimi¹,

Hermansson²,

Batchu³

et al. 2010

Journal of Biological Chemistry

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To better understand the principles underlying the substrate specificity of A-type phospholipases (PLAs), a high throughput mass spectrometric assay was employed to study the effect of acyl chain length and unsaturation of phospholipids on their rate of hydrolysis by three different secretory PLAs in micelles and vesicle bilayers. With micelles, each enzyme responded differently to substrate acyl chain unsaturation and double bond position, probably reflecting differences in the accommodative properties of their substrate binding sites. Experiments with saturated acyl positional isomers indicated that the length of the sn2 chain was more critical than that of the sn1 chain, suggesting tighter association of the former with the enzyme. Only the first 9 -10 carbons of the sn2 acyl chain seem to interact intimately with the active site. Strikingly, no discrimination between positional isomers was observed with vesicles, and the rate of hydrolysis decreased far more with increasing chain length than with micelles, suggesting that translocation of the phospholipid substrate to the active site is rate-limiting with bilayers. Supporting this conclusion, acyl chain structure affected hydrolysis and spontaneous intervesicle transfer, which correlates with lipid efflux propensity, analogously. We conclude that substrate efflux propensity plays a more important role in the specificity of secretory PLA 2 s than commonly thought and could also be a key attribute in phospholipid homeostasis in which (unknown) PLA 2 s are key players.Type A phospholipases (PLAs) 3 are ubiquitous enzymes involved in many biological phenomena, such as signal transduction (1) and phospholipid homeostasis (2), including acyl chain remodeling (3) and degradation (4). However, the identities of the specific proteins involved in these phenomena have often not been established. PLAs are also clinically relevant because they have been implicated in many common diseases or disorders, including atherosclerosis, allergy, Alzheimer disease, and cancer (5).Several PLAs display significant specificity toward the phospholipid acyl chain(s) in vitro (6, 7). However, despite numerous studies, the factors underlying such specificity have remained largely elusive. In principle, two factors contribute to selective hydrolysis of phospholipids by PLA: 1) accommodation of the acyl chains in the protein lipid binding site and 2) the ease of efflux of the phospholipid substrate from the bilayer (8, 9). The understanding of acyl chain accommodation has been greatly hampered by the lack of crystal structures of PLAs complexed with a physiological substrate. Crystal structures have been obtained for some PLAs with a bound noncleavable shortchain substrate analogue (10 -13), but because these analogues have truncated acyl chains, they only provide very limited information on the factors underlying acyl chain specificity. Furthermore, the information could also be biased, since studies with other lipid-binding proteins have shown that the mode of accommodation of an alkyl...

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confidence: 99%

Substrate Efflux Propensity Plays a Key Role in the Specificity of Secretory A-type Phospholipases

Haimi¹,

Hermansson²,

Batchu³

et al. 2010

Journal of Biological Chemistry

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“…In using this assay we observed complete linearity at least up to 10 mg of I or II even in the presence of fluorescent derivatives of C 6 or C 12 fatty acids. The slower hydrolysis of C 6 -NBD-PC (or C 6 -NBDbPC) as compared to C 12 -NBD-PC (or C 12 -NBD-bPC), may be attributed to poor binding of the substrate to phospholipase A 2 [6,7]. It appears that the observed differences between the hydrolysis rates of I and II are not due to differences between the physical states of these two types of phospholipid but could arise from differences between the structures of these lipids in the mixed micelles.…”

Section: Discussionmentioning

confidence: 99%

“…It was treated with N. naja snake venom (Sigma) and the resulting 1-hexadecanoyloxy-3-hydroxy-(3R)-but-4-yl-[2-(trimethylammonium)ethyl] phosphate was isolated in pure form by following the published procedure [10]. Reaction of this lysolipid with 6-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) aminohexanoic acid or 12-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) aminododecanoic acid in the presence of 1,1 H -carbonyldiimidazole [7] Fig. 1, C 12 -NBD-PC) were prepared using 1-hexadecanoyl-sn-glycero-3-phosphocholine as the starting material, which in turn was prepared as described earlier [11].…”

Section: General Methodsmentioning

confidence: 99%

“…To it was added Tris/HCl (25 mm, pH 8.5) containing 4.25 mm Triton X-100, 100 mm KCl and 10 mm Ca 2+ [6,7]. The suspension was sonicated untill a clear solution was obtained, and incubated for 5 min at 30 8C.…”

Section: Phospholipase a 2 -Catalysed Hydrolysismentioning

confidence: 99%

“…Apart from the sn-2 acyl chain [5], the acyl chain at the sn-1 position also plays an important role in phospholipase A 2 ±glycerophos-pholipid interactions [6,7]. It has been reported that an increase in the hydrophobicity of the sn-1 acyl residue increases the affinity between the phospholipid molecule and the enzyme [6].…”

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Probing the role of C‐1 ester group in Naja naja phospholipase  A₂–phospholipid interactions using butanetriol‐containing  phosphatidylcholine analogues

Puri

Arora

Gupta

1999

European Journal of Biochemistry

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To understand the role of the ester moiety of the sn-1 acyl chain in phospholipase A 2 ±glycerophospholipid interactions, we introduced an additional methylene residue between the glycerol C1 and C2 carbon atoms of phosphatidylcholines, and then studied the kinetics of hydrolysis and the binding of such butanetriol-containing phospholipids with Naja naja phospholipase A 2 . Hydrolysis was monitored by using phospholipids containing a NBD-labelled sn-2 acyl chain and binding was ascertained by measuring the protein tryptophan fluorescence. The hydrolysis of butanetriol-containing phospholipids was invariably slower than that of the glycerol-containing phospholipids. In addition, the enzyme binding with the substrate was markedly decreased upon replacing the glycerol residue with the 1,3,4-butanetriol moiety in phosphatidylcholines. These results have been interpreted to suggest that the sn-1 ester group in glycerophospholipids could play an important role in phospholipase A 2 ± phospholipid interactions.Keywords: phospholipase A 2 ; phosphatidylcholine; phospholipid conformation; butanetriol analogue.Cobra venom phospholipase A 2 is a Ca 2+ -dependent enzyme that catalyses the hydrolysis of the fatty acyl group at the sn-2 position of membrane phospholipids. It is composed of a single polypeptide chain of 119 amino acids which contains seven disulphide bonds [1]. The crystal structures of phospholipase A 2 from several sources have been determined [1±3], and the amino acids that form the enzyme catalytic site have been reported to be highly conserved [1]. Also, the structure of the catalytic surface has been shown to be exactly identical regardless of the degree of evolutionary divergence, state of oligomerization, ionic strength, or pH of the crystallization conditions [3,4].Binding of phospholipase A 2 with phosphatidylcholines involves interactions of the enzyme active site calcium with the phospholipid phosphate oxygen and the sn-2 carbonyl oxygen followed by the hydrophobic interactions between the phospholipid fatty acyl chains and the enzyme catalytic site [2±5]. Apart from the sn-2 acyl chain [5], the acyl chain at the sn-1 position also plays an important role in phospholipase A 2 ±glycerophos-pholipid interactions [6,7]. It has been reported that an increase in the hydrophobicity of the sn-1 acyl residue increases the affinity between the phospholipid molecule and the enzyme [6]. However, it is not yet known whether the ester moiety (-CO-O-) of the sn-1 acyl chain plays any role in phospholipase A 2 ± glycerophospholipid interactions. To investigate this we introduced one additional methylene residue between the glycerol C1 and C2 carbon atoms and then studied the interactions of the resulting modified phospholipids (Fig. 1) with Naja naja phospholipase A 2 . Results of these studies indicate that the sn-1 ester moiety plays an important role in binding of the enzyme with the substrate. MATERIALS AND METHODS MaterialsPhospholipase A 2 from N. naja snake venom, sn-glycero-3-phosphocholine, 6-N-(7-nitrobe...

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Continuous, Vesicle-Based Fluorimetric Assays of 14- and 85-kDa Phospholipases A2

Bayburt

Street

et al. 1995

Analytical Biochemistry

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Defining the dimensions of the catalytic site of phospholipase A2 using amide substrate analogs

Cited by 39 publications

References 1 publication

Substrate Efflux Propensity Plays a Key Role in the Specificity of Secretory A-type Phospholipases

Substrate Efflux Propensity Plays a Key Role in the Specificity of Secretory A-type Phospholipases

Probing the role of C‐1 ester group in Naja naja phospholipase  A₂–phospholipid interactions using butanetriol‐containing  phosphatidylcholine analogues

Continuous, Vesicle-Based Fluorimetric Assays of 14- and 85-kDa Phospholipases A2

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Defining the dimensions of the catalytic site of phospholipase A2 using amide substrate analogs

Cited by 39 publications

References 1 publication

Substrate Efflux Propensity Plays a Key Role in the Specificity of Secretory A-type Phospholipases

Substrate Efflux Propensity Plays a Key Role in the Specificity of Secretory A-type Phospholipases

Probing the role of C‐1 ester group in Naja naja phospholipase A2–phospholipid interactions using butanetriol‐containing phosphatidylcholine analogues

Continuous, Vesicle-Based Fluorimetric Assays of 14- and 85-kDa Phospholipases A2

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Probing the role of C‐1 ester group in Naja naja phospholipase  A₂–phospholipid interactions using butanetriol‐containing  phosphatidylcholine analogues