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The Escherichia coli outer-membrane phospholipase A (OM PLA) is a membrane-bound acyl hydrolase with a broad substrate specificity. In order to obtain more insight into the mechanism of action of this enzyme, we designed an active-site-directed inhibitor for OM PLA on the basis of the known substrate specificity as a first step in the elucidation of the catalytic mechanism of this enzyme. The inhibitor, hexadecanesulfonyl fluoride, consists of a long hydrocarbon chain for high-affinity binding by the enzyme and a sulfonyl fluoride moiety as a reactive group. The kinetics of the inactivation of OM PLA by hexadecanesulfonyl fluoride were studied in Triton X-100 micelles. Inactivation is very fast, specific and shows the same characteristics with respect to acyl specificity, pH profile and metal ion requirement as the activity of OM PLA on substrates. Incubation of OM PLA with a stoichiometric amount of hexadecanesulfonyl fluoride leads to a total and irreversible loss of enzyme activity, resulting from the sulfonylation of Ser144. This Ser144, which we suggest to be the active-site serine of OM PLA, is part of the sequence HDSNG, whereas in the water-soluble serine proteases and lipases the structural motif GXSXG is normally encountered. On the basis of the kinetics of inactivation of OM PLA by hexadecanesulfonyl fluoride, we discuss a possible catalytic mechanism of the enzyme. [ABSTRACT FROM AUTHOR]

The fluorescent probe 8-anilinonaphtalene-1-sulfonate (ANS) binds at the active site of the Naja melanoleuca snake venom phospholipase A2, thus protecting the enzyme against active-site-directed chemical modification. Both hydrophobic and electrostatic interactions are involved in the binding. At pH 7.5, a binding constant of 100 µM was determined, which improved twofold upon addition to the enzymatic cofactor Ca2+. The pH dependence of the ANS binding in the absence and presence of Ca2+ ions showed a perturbation of a group with a pKa value of 5.2, which could be assigned to the carboxylate group of the Ca2+-binding ligand Asp49 at the active site of the protein. Monomeric concentrations of the substrate analog n-decylphosphocholine displace ANS from the protein, indicating again that both ligands bind at the active site. Binding studies with several modified N. melanoleuca enzymes showed that a loss of enzymatic activity on aggregated substrates was correlated with a loss of affinity for the active site bound ANS molecule. It is suggested therefore, that the fluorescent ANS probe can detect structural rearrangements at the active site, which are important for enzymatic activity. [ABSTRACT FROM AUTHOR]

In phospholipase A2 from Naja melanoleuca snake venom all tour lysines were converted into the ε-amidinated derivatives without reaction of the α-amino group. The amidinated phospholipase (AMPA) showed high enzymatic activity. Starting from AMPA, chemical modification reactions were carried out at the α-amino function. This group was blocked with a tert-butyloxycarbonyt or a phenylthiocarbamyl group. Furthermore the polypeptide chain was shortened by one residue by removing the N-terminal asparagine, resulting in the formation of des-Asn1-AMPA. The native enzyme was shortened by eight residues by cyanogen bromide cleavage at the single methionine8 residue. Although all modified proteins show a reduced affinity for monomeric lipids, they are easily saturated with micellar substrate analogs. Whereas the removal of the N-terminal octapeptide abolished all enzymatic activity the other modified enzymes possess a low (1%), but measurable enzymatic activity. It is concluded that chemical modifications in the N-terminal region give rise to a distortion of the active site, thus reducing the activity of the lipid-bound enzyme. [ABSTRACT FROM AUTHOR]

Carboxylase groups in bovine pancreatic phospholipase A2 were modified using a water-soluble carbodiimide and semicarbazide. At pH 5.5 rapid modification of 13 out of the total of 15 carboxylates occurred, leaving Asp-39 and Asp-99 unmodified. Subsequent modification with radioactive semicarbazide at pH 3.5 resulted in the labelling of Asp-39. In the modified proteins both Ca2+ binding and enzymatic activity are completely lost. Modification of the enzyme at pH 5.5 in the presence of Ca2+ ions yielded a protein with three unmodified carboxylates which is capable of binding Ca2+ ions, while 15% residual activity was found. A second reaction, using [14C]semicarbazide at pH 5.5 in the absence of Ca2+, leads to the incorporation of one mole of [14C]semicarbazide/mole of protein with loss of enzymatic activity and Ca2+ binding. The label was found to be attached to Asp-49, demonstrating the essential role of Asp-49 in Ca2+ binding. Studies on the pH dependence of the Ca2+ binding to the protein suggested that Asp-49 has an apparent pK of 5.2. [ABSTRACT FROM AUTHOR]

Some highly purified phospholipases A from the venom of viperidae, crotalidae and elapidae were found to have anticoagulant properties. All phospholipases which exhibited anticoagulant properties are characterized by a high isoelectric point, but not all strongly basic phospholipases are anticoagulant. Anticoagulant phospholipases hydrolyse highly packed monomolecular films of phospholipids without any lag time while non-anticoagulant phospholipases present considerable induction times indicative of a tow penetrating power. When the ester linkages in the procoagulant lipids were replaced by the non-hydrolysable ether bonds, the mixture retained its clotting ability even in the presence of phospholipases, thus suggesting that anticoagulant phospholipases prevent clot formation by hydrolysis of phospholipids. This was confirmed by chemical modification of phospholipases, viz. alkylation of the active-centre histidine with 1-bromo-octan-2-one. This modification yielded proteins which had lost their anticoagulant properties but which retained a high affinity for phospholipids. [ABSTRACT FROM AUTHOR]

The complete amino acid sequence of bovine phospholipase A2 (EC 3.1.1.4) was determined. This enzyme has a molecular weight of 13 782 and consists of a single polypeptide chain of 123 amino acids cross-linked by seven disulfide bridges. The main fragmentation of the polypeptide chain was accomplished by digesting the reduced and thialaminated derivative of the protein with trypsin, staphylococcal protease and cyanogen bromide. A number of chymotryptic peptides were used for alignment and to obtain overlaps of at least two residues. The sequence of the peptides was determined by Edman degradation by means of direct phenylthiohydantoin identification in combination with identification as dansyl amino acids. Although 71% of all residues of phospholipase A2 from bovine, porcine and equine sources are conserved, bovine phospholipase A2 differs from the others by the total number of residues and by substitutions at 20 (porcine) and 33 (equine) positions. [ABSTRACT FROM AUTHOR]

The genes coding for the mature part of the lipases from Staphylococcus aureus NCTC8530 and Staphylococcus hyicus have been cloned and overexpressed in Escherichia coli as fusion proteins with an N-terminal hexa-histidine tag The enzymes accumulated in the cytoplasm and were purified using sequential precipitation with protamine sulphate and ammonium sulphate, followed by metal-affinity and hydroxyapatite chromatography. The yield of pure lipase was 4.5 mg/g wet cells for S. aureus lipase and 13 mg/g for S hyicus lipase. The purified enzymes need calcium for activity, albeit with different affinities, and a low residual activity was found in the absentee of calcium. In contrast to S. hyicuss lipase, not only strontium but also barium can replace calcium with full retention of activity of S, aureus lipase. Whereas S. hyicus lipase is optimally active at pH 8.5, the optimum pH for enzymatic activity for S. aureus lipase was found to be pH 6.5. The S. aureus lipase has a narrow substrate specificity: short-chain triacylglycerols and acyl esters of both p-nitrophenol and umbelliferone are readily degraded, whereas medium- and long-chain lipids, as well as phospholipids, are poor substrates. In contrast, S. hyicus lipase prefers phospholipids as substrate and hydrolyses neutral lipids irrespective of their chain length. The results are discussed in view of the large sequence similarity between both lipases. [ABSTRACT FROM AUTHOR]

Blood platelets play a central role in haemostasis by leading to plug formation and by increasing the efficiency of blood coagulation. We have previously shown that blood platelets contain a group II secretory phospholipase A2 (sPLA2 grII) which is released into the extracellular medium upon activation but is unable to stimulate blood platelets. We presently reported an investigation of the putative involvement of the human sPLA2 grII (hsPLA2 grII) in the coagulation process, both in the absence and in the presence of activated platelets. We show that this enzyme prolongs the recalcification time of blood plasma even in the presence of activated platelets. The positive action of blood platelets on coagulation is correlated, at least in part, with the appearance at the cellular surface of procoagulant phospholipids which constitute a potential target for hsPLA2 grII. We therefore investigated the involvement of its enzymatic activity in the anticoagulant effect of this enzyme. We observed that the replacement of CaCl2 by SrCl2 to initiate the coagulation cascade did not suppress, but rather increased, the inhibitory action of hsPLA2 grII. Moreover, hsPLA2 grII hydrolyzed only a minor proportion of platelet phospholipids, and it did not affect plasma phospholipids. Taken together, these observations strongly suggegst that the major action of hsPLA2 grII on blood coagulation does not involve the hydrolysis of phospholipids, in contrast with the strong anticoagulant effect of the group II venom phospholipase A2 from Crotalus durrissus terrificus. We next studied which step of the coagulation cascade was affected by hsPLA2 grII. Using purified coagulation factors, we demonstrated that hsPLA2 grII strongly inhibited the prothrombinase activity. This inhibitory effect was independent of the presence of phospholipids but required factor Va, leading to the hypothesis that hsPLA2 grII inhibited this factor. Further, the anticoagulation effect of hsPLA2 grII was observed on normal and factor-X-deficient plasma, but not on factor-V-deficient plasma. In conclusion, the anticoagulation action of hsPLA2 grII is based on a nonenzymatic mechanism of action involving the inhibition of factor Va. [ABSTRACT FROM AUTHOR]

Escherichia coli outer-membrane phospholipase A (OMPLA) is thought to be a member of the class of serine hydrolases, having a classical Asp-His-Ser catalytic triad [Horrevoets, A. J. G., Verheij, H. M. & de Haas, G. H. (1991) Eur. J. Biochem. 198-247–253]. To identify the histidine residue that is important for catalytic activity, the four histidine residues in E. coli OMPLA that are conserved in other enterobacterial OMPLA enzymes were replaced by cysteine residues using PCR-directed, site-specific mutagenesis. The resulting mutant proteins were all well expressed and displayed heat modifiability, indicating that they were properly folded. Enzyme assays showed that only the His142Cys mutant protein was lacking enzymatic activity. In addition, a His142Gly mutant protein appeared to be inactive. These results show that His142 is important for the enzymatic activity of OMPLA. [ABSTRACT FROM AUTHOR]

Porcine pancreatic phospholipase A2 (PLA2) was studied by site-directed mutagenesis. Arg53 and/or Lys56 were replaced by a methionine (R53M or K56M, respectively) in combination with the Tyr69→Phe (Y69F) substitution. These substitutions improved the activity on micellar and monomeric zwitterionic substrates and reduced the activity on negatively charged substrates compared to the Y69F mutant. With the neutral substrate 1,2-didodecanoyl-sn-glycerol-3-dimethyl phosphate (Lau2GroMe2P) a 20-fold increase of activity was observed for the 69F53M56M mutant, whereas this mutant showed a lower activity than native PLA2 on zwitterionic substrates. Thus the ratio Lau2GroMe2P/Lau2GroPCho has become 65 times higher for 69F53M56M compared to native phospholipase A2, illustrating that the substrate specificity has changed enormously. The methionine substitutions were also prepared in a 69F mutant in which a part of the surface loop (residues 62–66) was deleted. Also in this deletion mutant these substitutions showed a similar effect as the substitutions in the native 69F mutant. Furthermore it was shown that deletion of the loop increases the activity on micellar lecithins and negatively charged micellar substrates, but reduces the activity on Lau2GroMe2P. Therefore it can be concluded that the loop is important for the recognition of substrates. We also show that the loop plays a role in the dimerization of these proteins. Dimerization may account for the high activities observed for some mutants acting on monomeric substrate. [ABSTRACT FROM AUTHOR]

Presents a comparative biochemical and molecular analysis of the Staphylococcus hyicus, Staphylococcus aureus and a hybrid lipase. Indication for a C-terminal phospholipase domain; Determination of the transcriptional start site; Expression of the lipase gene in the protease-negative Staphylococcus carnosus.

Both methionine residues in phospholipase A2 (PLA2) from porcine pancreas have been replaced by leucines with retention of full enzymatic activity. The methionine-less mutant has been expressed as a Cro-LacZ fusion protein in Escherichia coli, from which a pro-PLA2 was liberated by chemical cleavage with CNBr. The general applicability of CNBr cleavage of proteins lacking mcthionine residue(s) was demonstrated by replacing the single Met8 in human platelet phospholipase A2 (HPPLA2) by a leucine residue, and the introduction of a methionine at a position just preceding the HPPLA2 sequence. This protein was expressed in E. coil as a 68-kDa Cro-LacZ fusion protein. CNBr cleavage liberated the HP-PLA2 fragment which was reoxidized in vitro. The [Met8→ Leu]HP-PLA2 is monomeric in aqueous solutions, requires calcium ions in the millimolar range for enzymatic activity and has optimal activity around pH 8, ρ-Bromophenacyl bromide rapidly inactivates the enzyme with calcium ions having a protective effect. The highest specific activities, 2400 U/mg and 9300 U/mg, were found with pure micelles of 1,2-dioctanoyl-sn-glycero-3-phosphoglycol and with mixed micelles of taurodeoxycholate and 1,2-dioctanoyl-sn-glycero-3-phosphoglycol, respectively. In mixed micelles the activity on dioleoyl phospholipids decreases in the order phosphatidylglycerol > phosphatidylethanolamine » phosphatidylcholine. The enzyme has low activity on monomeric 1,2diheptanoyl-sn-glycero-3-phosphocholine as a substrate, but high activity on micelles with a distinct jump in activity at the critical micellar concentration. The binding of the HP-PLA2, porcine pancreatic PLA2 and PLA2 from Naja melanoleuca venom to lipid/water interfaces was determined with micellar solutions of the substrate analog n-hexadecylphosphocholine. The HP-PLA2 has a high apparent Kd (2 mM) compared to pancreatic (0.2 mM) and venom (0.03 mM) PLA2. In mixed micelles of taurodeoxycholate and 1,2-didodecanoyl-sn-glycero-3-phosphocholine, the competitive inhibition of HP-PLA2 by the R and S enantiomers of 2-tetradecanoylaminohexanol-l-phosphocholine, its phosphoglycol, and its phosphoethanolamine derivatives were tested. The S enantiomers are only weak inhibitors, whereas the R enantiomers are potent inhibitors. The inhibitory power depends on the nature of the polar head group and increases in the order phosphocholine « phosphoethanolamine < phosphoglycol. The best inhibitor, (R)-2-tetradecanoylaminohexanol-l-phosphoglycol, binds 2200 times stronger than the substrate to the HP-PLA2 active site. [ABSTRACT FROM AUTHOR]

The activity of the Escherichia coli outer-membrane phospholipase (OM PLA) is strictly regulated in its natural habitat, the E. coli outer membrane. OM PLA can be reconstituted in phospholipid bilayers, resulting in low specific activity of the enzyme compared to its activity on mixed lipid/detergent micelles. The enzyme can be activated by the addition to these vesicles of the membrane-perturbing peptides polymyxin B, melittin or cardiotoxin resulting in hydrolysis of mainly the sn-1 ester bond of the phospholipids as is also observed in vivo. We used the affinity label hexadecanesulfonyl fluoride to probe the influence of lipid environment on the activity of OM PLA. In detergent and substrate micelles, the rate constant for the sulfonylation of the active-center serine of the purified OM PLA by the affinity label hexadecanesulfonyl fluoride depends on amphiphile concentration. We have reported a similar influence of amphiphile concentration on the activity of the enzyme [Horrevoets, A. J. G. et al. (1989) Biochemistry 28, 1139-1147]. Analysis of the rates of inactivation of OM PLA by hexadecanesulfonyl fluoride in vesicles composed of various phospholipids indicated that activation of the enzyme by membrane-perturbing peptides can be accurately quantified with this affinity label. Our results show that the affinity label hexadecanesulfonyl fluoride can be used to monitor the state of activation of OM PLA in different lipid environments, including non-hydrolyzable substrate analogues. Implications for the in vivo situation are discussed. [ABSTRACT FROM AUTHOR]

In addition to the Ca2+ ion at the active site, porcine pancreatic phospholipase A2 (PLA) is known to bind a second calcium ion with a lower affinity at alkaline pH. The second calcium-binding site has been held responsible for effective interaction of phospholipase with organized lipid/water interfaces [van Dam-Mieras, M. C. E., Slotboom, A. J., Pieterson, W. A. and de Haas, G. H. (1975) Biochemistry 14, 5387-5394]. To study the identity of the acidic amino acid residues involved in liganding the second calcium ion in detail, we used site-directed mutagenesis to specifically alter the cDNA encoding porcine pancreatic phospholipase. Three mutant phospholipase species were constructed, each of which lacked one of the potentially important carboxylates: Asp66→Asn, Glu71→Asn and Glu92→Gln. The Gln92 mutant PLA displayed the same properties as native phospholipase indicating that Glu92 is not important for binding the second metal ion. However, Glu71 and, to a lesser extent, Asp66 are both directly involved in the low-affinity calcium binding. [ABSTRACT FROM AUTHOR]

In order to probe the role of Asp-49 in the active site of porcine pancreatic phospholipase A2 two mutant proteins were constructed containing either Glu or Lys at position 49. Their enzymatic activities and their affinities for substrate and for Ca2+ ions were examined in comparison with the native enzyme. Enzymatic characterization indicated that the presence of Asp-49 is essential for effective hydrolysis of phospholipids. Conversion of Asp-49 to either Glu or Lys strongly reduces the binding of Ca2+ ions in particular for the lysine mutant but the affinity for substrate analogues is hardly affected. Extensive purification of [Lys49]phospholipase A2 from the venom of Agkistrodon piscivorus piscivorus yielded a protein which was 4000 times less active than the basic [Asp49]phosphotipase A2 from this venom. Inhibition studies with p-bromophenacyl bromide showed that this residual activity was due to a small amount of contaminating enzyme and that the Lys-49 homologue itself is inactive. The results obtained both with the porcine pancreatic phospholipase A2 mutants and with the native venom enzymes show that Asp-49 is essential for the catalytic action of phospholipase A2. [ABSTRACT FROM AUTHOR]

The cDNA coding for porcine pancreatic prophospholipase A2 (proPLA) has been cloned and expressed in Saccharomyces cerevisiae. Expression and secretion of proPLA could only be obtained after fusing the proPLA to the prepro sequence of the yeast α-mating factor. Upon secretion, the fusion protein was cleaved by the KEX2 protease yielding a 140-amino-acid zymogen-like form of the phospholipase A2. This protein was purified in high yield by ion-exchange chromatography. Limited proteolysis with trypsin cleaved the `zymogen' to yield active phospholipase A2, which was indistinguishable from the authentic porcine pancreatic enzyme. These results show that a protein with a disulphide bridge content as high as 7 per 124 amino acid residues can be correctly processed by the yeast secretory apparatus. [ABSTRACT FROM AUTHOR]

Carboxylase groups in bovine pancreatic phospholipase A2 were modified using a water-soluble carbodiimide and semicarbazide. At pH 5.5 rapid modification of 13 out of the total of 15 carboxylates occurred, leaving Asp-39 and Asp-99 unmodified. Subsequent modification with radioactive semicarbazide at pH 3.5 resulted in the labelling of Asp-39. In the modified proteins both Ca2+ binding and enzymatic activity are completely lost. Modification of the enzyme at pH 5.5 in the presence of Ca2+ ions yielded a protein with three unmodified carboxylates which is capable of binding Ca2+ ions, while 15% residual activity was found. A second reaction, using [14C]semicarbazide at pH 5.5 in the absence of Ca2+, leads to the incorporation of one mole of [14C]semicarbazide/mole of protein with loss of enzymatic activity and Ca2+ binding. The label was found to be attached to Asp-49, demonstrating the essential role of Asp-49 in Ca2+ binding. Studies on the pH dependence of the Ca2+ binding to the protein suggested that Asp-49 has an apparent pK of 5.2. [ABSTRACT FROM AUTHOR]

In addition to the Ca2+ ion at the active site, porcine pancreatic phospholipase A2 (PLA) is known to bind a second calcium ion with a lower affinity at alkaline pH. The second calcium-binding site has been held responsible for effective interaction of phospholipase with organized lipid/water interfaces [van Dam-Mieras, M. C. E., Slotboom, A. J., Pieterson, W. A. and de Haas, G. H. (1975) Biochemistry 14, 5387-5394]. To study the identity of the acidic amino acid residues involved in liganding the second calcium ion in detail, we used site-directed mutagenesis to specifically alter the cDNA encoding porcine pancreatic phospholipase. Three mutant phospholipase species were constructed, each of which lacked one of the potentially important carboxylates: Asp66→Asn, Glu71→Asn and Glu92→Gln. The Gln92 mutant PLA displayed the same properties as native phospholipase indicating that Glu92 is not important for binding the second metal ion. However, Glu71 and, to a lesser extent, Asp66 are both directly involved in the low-affinity calcium binding. [ABSTRACT FROM AUTHOR]

In order to probe the role of Asp-49 in the active site of porcine pancreatic phospholipase A2 two mutant proteins were constructed containing either Glu or Lys at position 49. Their enzymatic activities and their affinities for substrate and for Ca2+ ions were examined in comparison with the native enzyme. Enzymatic characterization indicated that the presence of Asp-49 is essential for effective hydrolysis of phospholipids. Conversion of Asp-49 to either Glu or Lys strongly reduces the binding of Ca2+ ions in particular for the lysine mutant but the affinity for substrate analogues is hardly affected. Extensive purification of [Lys49]phospholipase A2 from the venom of Agkistrodon piscivorus piscivorus yielded a protein which was 4000 times less active than the basic [Asp49]phosphotipase A2 from this venom. Inhibition studies with p-bromophenacyl bromide showed that this residual activity was due to a small amount of contaminating enzyme and that the Lys-49 homologue itself is inactive. The results obtained both with the porcine pancreatic phospholipase A2 mutants and with the native venom enzymes show that Asp-49 is essential for the catalytic action of phospholipase A2. [ABSTRACT FROM AUTHOR]

Porcine pancreatic phospholipase A2 (PLA2) was studied by site-directed mutagenesis. Arg53 and/or Lys56 were replaced by a methionine (R53M or K56M, respectively) in combination with the Tyr69→Phe (Y69F) substitution. These substitutions improved the activity on micellar and monomeric zwitterionic substrates and reduced the activity on negatively charged substrates compared to the Y69F mutant. With the neutral substrate 1,2-didodecanoyl-sn-glycerol-3-dimethyl phosphate (Lau2GroMe2P) a 20-fold increase of activity was observed for the 69F53M56M mutant, whereas this mutant showed a lower activity than native PLA2 on zwitterionic substrates. Thus the ratio Lau2GroMe2P/Lau2GroPCho has become 65 times higher for 69F53M56M compared to native phospholipase A2, illustrating that the substrate specificity has changed enormously. The methionine substitutions were also prepared in a 69F mutant in which a part of the surface loop (residues 62–66) was deleted. Also in this deletion mutant these substitutions showed a similar effect as the substitutions in the native 69F mutant. Furthermore it was shown that deletion of the loop increases the activity on micellar lecithins and negatively charged micellar substrates, but reduces the activity on Lau2GroMe2P. Therefore it can be concluded that the loop is important for the recognition of substrates. We also show that the loop plays a role in the dimerization of these proteins. Dimerization may account for the high activities observed for some mutants acting on monomeric substrate. [ABSTRACT FROM AUTHOR]