UNIPROT:P00750 - FACTA Search

Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P00750 (PLA)
16,800 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Ammodytoxins (Atxs) are group II phospholipases A(2) (PLA(2)s) with presynaptic toxicity from venom of the snake Vipera ammodytes ammodytes. The molecular basis of their neurotoxicity, and that of similar PLA(2) toxins, is still to be explained. To address this problem, a surface-exposed aromatic residue, Phe(24), in the N-terminal region of the most potent Atx, AtxA, was replaced by other aromatic (tyrosine, tryptophan), hydrophobic (alanine) and polar uncharged (serine, asparagine) residues. The mutants were produced in the bacterial expression system, refolded in vitro and purified to homogeneity. All but the Trp(24) mutant, whose activity was similar to that of the wild type, showed a considerable decrease (40-80%) in enzymic activity on a micellar phosphatidylcholine substrate. This result indicates an important role for the aromatic side chains of phenylalanine or tryptophan, but not tyrosine, in PLA(2) activity, very likely at a stage of interfacial adsorption of the enzyme to zwitterionic aggregated substrates. The substitutions of Phe(24) also significantly decreased toxicity in mice, with the most prominent decrease, of 130-fold, observed in the case of the Asn(24) mutant. The results with the mutants show that there is no correlation between enzymic activity, lethality and binding affinity for three AtxA neuronal receptors (R180, R25 and calmodulin). Our results suggest a critical involvement of Phe(24) in the neurotoxicity of AtxA, apparently at a stage which does not involve the interaction with the known Atx-binding neuronal proteins and catalytic activity.
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PMID:Phenylalanine-24 in the N-terminal region of ammodytoxins is important for both enzymic activity and presynaptic toxicity. 1193 65

We had previously shown that the cholesterol esterification activity of lecithin:cholesterol acyltransferase (LCAT) is destroyed by oxidation, but still it retains the ability to hydrolyse water-soluble substrates. This suggested that the inactivation of the enzyme is not due to its catalytic function, but due to a loss of its hydrophobic binding. Since recent studies have shown that a tryptophan residue in the putative interfacial domain (Trp(61)) is critical for the activity, we determined the possible role of this residue in the oxidative susceptibility and substrate specificity of LCAT by site-directed mutagenesis. Deletion of Trp(61) resulted in a 56% loss of cholesterol esterification (LCAT) activity, but the phospholipase A(2) (PLA(2)) and the esterase activities of the enzyme were stimulated slightly. Replacing Trp(61) with another aromatic residue [Trp(61)-->Tyr (W61Y)] resulted in an increase in all activities (14-157%), whereas replacing it with an aliphatic residue [Trp(61)-->Gly (W61G)] caused a dramatic loss of LCAT (-90%) and PLA(2) (-82%) activities, but not the esterase activity (-5%). W61Y was the most sensitive to oxidation, whereas W61G was the most resistant, with respect to the LCAT and PLA(2) activities. However, the activities which do not involve interfacial binding, namely the esterase activity and the transesterification of short-chain phospholipids, were more resistant to oxidation in all LCATs, indicating a selective loss of the interfacial binding by oxidation. Furthermore, replacing the two cysteines (Cys(31) and Cys(184)) in the Trp(61) deletion mutant caused additional resistance of the enzyme to oxidizing agents, showing that both domains of the enzyme contribute independently to its oxidative susceptibility. Since the hydrolysis of truncated phospholipids, generated during the oxidation of low-density lipoproteins, does not require the interfacial-binding domain, our results suggest that LCAT may take part in the detoxification of these compounds even after the loss of its cholesterol esterification function.
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PMID:Role of the interfacial binding domain in the oxidative susceptibility of lecithin:cholesterol acyltransferase. 1196 70

The inhibition mechanism of serpins requires a change in structure to entrap the target proteinase as a stable acyl-enzyme complex. Although it has generally been assumed that reactive center loop insertion and associated conformational change proceeds in a concerted manner, this has not been demonstrated directly. Through the substitution of tryptophan with 7-azatryptophan and an analysis of transient reaction kinetics, we have described the formation of an inhibited serpin-proteinase complex as a single concerted transition of the serpin structure. Replacement of the four tryptophans of plasminogen activator inhibitor type-1 (PAI-1) with the spectrally unique analogue 7-azatryptophan permitted observations of conformational changes in the serpin but not those of the proteinase. Formation of covalent acyl-enzyme complexes, but not noncovalent Michaelis complexes, with tissue-type plasminogen activator (t-PA) or urokinase (u-PA) resulted in rapid decreases of fluorescence coinciding with insertion of the reactive center loop and expansion of beta-sheet A. Insertion of an octapeptide consisting of the P14-P7 residues of the reactive center loop into beta-sheet A produced the same conformational change in serpin structure measured by 7-azatryptophan fluorescence, suggesting that introduction of the proximal loop residues induces the structural rearrangement of the serpin molecule. The atom specific modification of the tryptophan indole rings through analogue substitution produced a proteinase specific effect on function. The reduced inhibitory activity of PAI-1 against t-PA but not u-PA suggested that the mechanism of loop insertion is sensitive to the intramolecular interactions of one or more tryptophan residues.
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PMID:A concerted structural transition in the plasminogen activator inhibitor-1 mechanism of inhibition. 1235

An important characteristic of the human group IIA secreted phospholipase A(2) (IIA PLA(2)) is the extremely low activity of this enzyme with phosphatidylcholine (PC) vesicles, mammalian cell membranes, and serum lipoproteins. This characteristic is reflected in the lack of ability of this enzyme to bind productively to zwitterionic interfaces. Part of the molecular basis for this lack of activity is an absence of tryptophan, a residue with a known preference for residing in the interfacial region of zwitterionic phospholipid bilayers. In this paper we have replaced the eight residues that make up the hydrophobic collar on the interfacial binding surface of the enzyme with tryptophan. The catalytic and interfacial binding properties of these mutants have been investigated, particularly those properties associated with binding to and hydrolysis of zwitterionic interfaces. Only the insertion of a tryptophan at position 3 or 31 produces mutants that significantly enhance the activity of the human IIA enzyme against zwitterionic interfaces and intact cell membranes. Importantly, the ability of the enzyme mutants to hydrolyze PC-rich interfaces such as the outer plasma membrane of mammalian cells was paralleled by enhanced interfacial binding to zwitterionic interfaces. The corresponding double tryptophan mutant (V3,31W) displays a specific activity on PC vesicles comparable to that of the human group V sPLA2. This enhanced activity includes the ability to interact with human embryonic kidney HEK293 cells, previously reported for the group V enzyme [Kim, Y. J., Kim, K. P., Rhee, H. J., Das, S., Rafter, J. D., Oh, Y. S., and Cho, W. (2002) J. Biol. Chem. 277, 9358-9365].
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PMID:Effect of tryptophan insertions on the properties of the human group IIA phospholipase A2: mutagenesis produces an enzyme with characteristics similar to those of the human group V phospholipase A2. 1280 88

Plasminogen activator inhibitor-1 (PAI-1) is a typical member of the serpin family that kinetically traps its target proteinase as a covalent complex by distortion of the proteinase domain. Incorporation of the fluorescently silent 4-fluorotryptophan analog into PAI-1 permitted us to observe changes in the intrinsic tryptophan fluorescence of two-chain tissue-type plasminogen activator (tPA) and the proteinase domain of tPA during the inhibition reaction. We demonstrated three distinct conformational changes of the proteinase that occur during complex formation and distortion. A conformational change occurred during the initial formation of the non-covalent Michaelis complex followed by a large conformational change associated with the distortion of the proteinase catalytic domain that occurs concurrently with the formation of stable proteinase-inhibitor complexes. Following distortion, a very slow structural change occurs that may be involved in the stabilization or regulation of the trapped complex. Furthermore, by comparing the inhibition rates of two-chain tPA and the proteinase domain of tPA by PAI-1, we demonstrate that the accessory domains of tPA play a prominent role in the initial formation of the non-covalent Michaelis complex.
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PMID:Distortion of the catalytic domain of tissue-type plasminogen activator by plasminogen activator inhibitor-1 coincides with the formation of stable serpin-proteinase complexes. 1450 Jul 31

Plasminogen activator inhibitor-1 (PAI-1) is a 43 kDa protein involved in the regulation of fibrinolysis. PAI-1 is the principal inhibitor of tissue-type plasminogen activator (t-PA), trapping the proteinase as an acyl-enzyme covalent complex (approximately 105 kDa). Four single tryptophan mutants of PAI-1 have been constructed in which three of the four tryptophan residues (Trp86, Trp139, Trp175, and Trp262) were replaced with phenylalanine. Biosynthetic incorporation of 5-fluorotryptophan (5F-Trp) into wild-type PAI-1 (5FW wtPAI-1) and the single tryptophan mutants (5FW86, 5FW139, 5FW175, and 5FW262) was achieved, allowing a (19)F NMR spectroscopic study of PAI-1 in its active and cleaved forms and in complex with t-PA. The (19)F NMR spectrum of active 5FW wtPAI-1 shows four clearly resolved peaks at -39.20, -49.26, -50.74, and -52.57 ppm relative to trifluoroacetic acid at 0 ppm. Unequivocal assignments of these four resonances in the spectrum of 5FW wtPAI-1 to specific tryptophan residues were accomplished by measuring the chemical shifts of the (19)F resonances of the single tryptophan mutants. There was close agreement between the resonances observed in 5FW wtPAI-1 and of those in the mutants for all three protein forms. This would imply little structural perturbation in the local structures of the tryptophan residues resulting from substitution by phenylalanine. The 5FW wtPAI-1 was observed to have lower second-order rate constant (k(app)) for the inhibition of t-PA than the natural tryptophan wtPAI-1, suggesting that the decreased activity may result from a small structural effect of the fluorine substituent of the indole ring. Further alterations in the k(app) and the stoichiometry of inhibition (SI) were observed in each of the mutants indicating an effect of the three tryptophan to phenylalanine mutations. Detailed interpretation of the (19)F NMR spectra of the PAI-1 mutants provides insights into the local segmental structure of the active form of the proteins and the structural changes that occur in the cleaved and t-PA complexed forms.
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PMID:19F NMR studies of plasminogen activator inhibitor-1. 1476 27

Association of class-II phospholipase A(2) (PLA(2)) with aggregated phospholipid substrate results in elevated levels of the Ca(2+)-dependent hydrolytic activity. The Asp49 residue participates in coordination of the Ca(2+) ion cofactor, however, in Lys49-PLA(2) homologues (Lys49-PLA(2)s), substitution of the Asp49 by Lys results in loss of Ca(2+) binding and lack of detectable phospholipid hydrolysis. Nevertheless, Lys49-PLA(2)s cause Ca(2+)-independent damage of liposome membranes. Bothropstoxin-I is a homodimeric Lys49-PLA(2) from the venom of Bothrops jararacussu, and in fluorescent marker release and dynamic light scattering experiments with DPPC liposomes we demonstrate activation of the Ca(2+)-independent membrane damaging activity by approximately 4 molecules of sodium dodecyl sulphate (SDS) per protein monomer. Activation is accompanied by significant changes in the intrinsic tryptophan fluorescence emission (ITFE) and near UV circular dichroism (UVCD) spectra of the protein. Subsequent binding of 7-10 SDS molecules results in further alterations in the ITFE and far UVCD spectra. Reduction in the rate of N-bromosuccinimide modification of Trp77 at the dimer interface suggests that initial binding of SDS to this region accompanies the activation of the membrane damaging activity. 1-anilinonaphthalene-8-sulphonic acid binding studies indicate that subsequent SDS binding to the active site is concomitant with the second structural transition. These results provide insights in the structural basis of amphiphile/protein coupling in class-II PLA(2)s.
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PMID:Activation of Ca2+-independent membrane-damaging activity in Lys49-phospholipase A2 promoted by amphiphilic molecules. 1532 39

A short peptide derived from the C-terminal region of Bothrops asper myotoxin II, a Lys49 phospholipase A(2) (PLA(2)), was previously found to reproduce the bactericidal activity of its parent molecule. In this study, a panel of eight PLA(2) myotoxins purified from crotalid snake venoms, including both Lys49 and Asp49-type isoforms, were all found to express bactericidal activity, indicating that this may be a common action of the group IIA PLA(2) protein family. A series of 10 synthetic peptide variants, based on the original C-terminal sequence 115-129 of myotoxin II and its triple Tyr-->Trp substituted peptide p115-W3, were characterized. In vitro assays for bactericidal, cytolytic and anti-endotoxic activities of these peptides suggest a general correlation between the number of tryptophan substitutions introduced and microbicidal potency, both against Gram-negative (Salmonella typhimurium) and Gram-positive (Staphylococcus aureus) bacteria. Peptide variants with high bactericidal activity also tended to be more cytolytic towards skeletal muscle C2C12 myoblasts, thus limiting their potential in vivo use. However, the peptide variant pEM-2 (KKWRWWLKALAKK) showed reduced toxicity towards muscle cells, while retaining high bactericidal potency. This peptide also showed the highest endotoxin-neutralizing activity in vitro, and was shown to functionally interact with lipopolysaccharide (LPS) using a chimeric bacteria model. The bactericidal and anti-endotoxic properties of pEM-2, combined with its relatively low toxicity towards eukaryotic cells, highlight it as a promising candidate for further evaluation of its antimicrobial potential in vivo.
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PMID:Antimicrobial activity of myotoxic phospholipases A2 from crotalid snake venoms and synthetic peptide variants derived from their C-terminal region. 1590 76

Simultaneous ablation of the two known activators of plasminogen (Plg), urokinase-type (uPA) and the tissue-type (tPA), results in a substantial delay in skin wound healing. However, wound closure and epidermal re-epithelialization are significantly less impaired in uPA;tPA double-deficient mice than in Plg-deficient mice. Skin wounds in uPA;tPA-deficient mice treated with the broad-spectrum matrix metalloproteinase (MMP) inhibitor galardin (N-[(2R)-2-(hydroxamido-carbonylmethyl)-4-methylpentanoyl]-L-tryptophan methylamide) eventually heal, whereas skin wounds in galardin-treated Plg-deficient mice do not heal. Furthermore, plasmin is biochemically detectable in wound extracts from uPA;tPA double-deficient mice. In vivo administration of a plasma kallikrein (pKal)-selective form of the serine protease inhibitor ecotin exacerbates the healing impairment of uPA;tPA double-deficient wounds to a degree indistinguishable from that observed in Plg-deficient mice, and completely blocks the activity of pKal, but not uPA and tPA in wound extracts. These findings demonstrate that an additional plasminogen activator provides sufficient plasmin activity to sustain the healing process albeit at decreased speed in the absence of uPA, tPA and galardin-sensitive MMPs and suggest that pKal plays a role in plasmin generation.
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PMID:Plasminogen activation independent of uPA and tPA maintains wound healing in gene-deficient mice. 1676 60

Phospholipase A(2) (PLA(2)) enzymes become activated by binding to biological membranes and hydrolyze phospholipids to free fatty acids and lyso-phospholipids, the precursors of inflammatory mediators. To understand the functional significance of amino acid residues at key positions, we have studied the effects of the substitution of Val(3) (membrane binding surface) and Phe(5) (substrate binding pocket) of human group IIA PLA(2) by tryptophan on the structure and function of the enzyme. Despite the close proximity of the sites of mutations, the V3W mutation results in substantial enhancement of the enzyme activity, whereas the F5W mutant demonstrates significantly suppressed activity. A structural analysis of all three proteins free in buffer and bound to membranes indicates that large differences in activities result from distinct conformational changes in PLA(2)s upon membrane binding. Although PLA(2) and the V3W mutant demonstrate a decrease in helical content and an increase in helix flexibility, the F5W mutant experiences partial distortion of the alpha-helical structure presumably resulting from the tendency of Trp(5) to insert into the membrane. Furthermore, whereas the PLA(2) and the V3W mutant bind to the membrane at similar and apparently productive-mode orientation, the F5W mutant binds to membranes with a distinctly different orientation. It is suggested that both the stimulatory effect of the V3W mutation and the inhibitory effect of the F5W mutation result from the high affinity of Trp for the membrane-water interface. Although Trp(3) at the membrane binding face of PLA(2) facilitates the proper membrane binding of the enzyme, Trp(5) in the internal substrate binding site causes partial unwinding of the N-terminal helix in order to interact with the membrane.
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PMID:Structural and functional effects of tryptophans inserted into the membrane-binding and substrate-binding sites of human group IIA phospholipase A2. 1702

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