sn-1,2-diacylglycerol kinase of Escherichia coli. Diacylglycerol analogues define specificity and mechanism

A detailed structure/function analysis of the substrate specificity of Escherichia coli sn-1,2-diacylglycerol kinase was performed with three goals in mind: (a) to define the substrate specificity; (b) to discover inhibitors; and (c) to elucidate the specificity of diacylglycerol-dependent inactivation. Forty-seven structural analogues of sn-1,2-diacylglycerol were prepared and examined as substrates, inhibitors, and irreversible inactivators of the enzyme using mixed micellar assay methods. Modification of the acyl chains or the sn-2 ester affected the apparent Km but had only small effects on Vm; modifications of the sn-1 ester, sn-3 methylene, or sn-3 hydroxyl had large effects on the apparent Vm and smaller effects on Km. Consistent with these observations, diacylglycerol analogues modified only in the acyl chains or sn-2 ester were not diacylglycerol kinase inhibitors, whereas analogues with substitutions of the sn-1 ester or sn-3 hydroxyl frequently caused inhibition. A hydrogen bond-donating group was required for an analogue to be a diacylglycerol kinase inhibitor. Studies of diacylglycerol kinase inactivation by the various analogues were consistent with the previous conclusion that this process involves an interaction of diacylglycerols with an enzyme conformation different from that active in catalysis (Walsh, J. P., and Bell, R. M. (1986) J. Biol. Chem. 261, 15062-15069). Studies with a water-soluble diacylglycerol, sn-1,2-dibutyrylglycerol, allowed direct comparison of diacylglycerol kinase activity in mixed micelles with that in native membranes. The results are discussed in relation to the structural requirements of other diacylglycerol-dependent enzymes.     

Acyloxyacyl hydrolase, a leukocyte enzyme that deacylates bacterial lipopolysaccharides, has phospholipase, lysophospholipase, diacylglycerollipase, and acyltransferase activities in vitro.

Human acyloxyacyl hydrolase (AOAH) is a leukocyte enzyme that hydrolyzes acyloxyacyl bonds in the lipid A region of bacterial lipopolysaccharide (LPS), thereby detoxifying the LPS. We report here that the enzyme also acts in vitro on glycerophospholipids, lysophospholipids, and diacylglycerol. While AOAH preferentially removes palmitate or stearate from the sn-1 position of phospholipid and diacylglycerol substrates that have unsaturated acyl chains in the sn-2 position, it is able to cleave both palmitates from sn-1,2-dipalmitoylphosphatidylcholine and sn-1,2-dipalmitoylglycerol. This apparent preference for removing saturated (or shorter) acyl chains from glycerolipids is consistent with its ability to cleave laurate more rapidly than palmitoleate from lipopolysaccharide (Erwin, A. L., and Munford, R. S. (1990) J. Biol. Chem. 265, 16444-16449). AOAH also catalyzes acyl transfer from LPS and phosphatidylethanolamine to acceptor lipids; approximately equal amounts of laurate and myristate are transferred from LPS to monooleoylglyceryl ether, forming acyloleoylglyceryl ether. The demonstration that AOAH has phospholipase, lysophospholipase, diacylglycerol lipase, and acyltransferase activities in vitro suggests that the enzyme may have roles in addition to LPS deacylation (detoxification) in phagocytic cells.     

Role of conserved active site residues in catalysis by phospholipase B1 from Cryptococcus neoformans

Phospholipase B1 (PLB1), secreted by the pathogenic yeast Cryptococcus neoformans, has an established role in virulence. Although the mechanism of its phospholipase B, lysophospholipase, and lysophospholipase transacylase activities is unknown, it possesses lipase, subtilisin protease aspartate, and phospholipase motifs containing putative catalytic residues S146, D392, and R108, respectively, conserved in fungal PLBs and essential for human cytosolic phospholipase A2 (cPLA2) catalysis. To determine the role of these residues in PLB1 catalysis, each was substituted with alanine, and the mutant cDNAs were expressed in Saccharomyces cerevisiae. The mutant PLB1s were deficient in all three enzymatic activities. As the active site structure of PLB1 is unknown, a homology model was developed, based on the X-ray structure of the cPLA2 catalytic domain. This shows that the two proteins share a closely related fold, with the three catalytic residues located in identical positions as part of a single active site, with S146 and D392 forming a catalytic dyad. The model suggests that PLB1 lacks the "lid" region which occludes the cPLA2 active site and provides a mechanism of interfacial activation. In silico substrate docking studies with cPLA2 reveal the binding mode of the lipid headgroup, confirming the catalytic dyad mechanism for the cleavage of the sn-2 ester bond within one of two separate binding tracts for the lipid acyl chains. Residues specific for binding arachidonic and palmitic acids, preferred substrates for cPLA2 and PLB1, respectively, are identified. These results provide an explanation for differences in substrate specificity between lipases sharing the cPLA2 catalytic domain fold and for the differential effect of inhibitors on PLB1 enzymatic activities.     

Partial purification of a lipolytic enzyme from Escherichia coli.

An enzyme with phospholipase Al activity was purified some 500-fold from Escherichia coli cell homogenates. Lipase, phospholipase A2, and lysophospholipase copurified with phospholipase A1 and the four activities displayed similar susceptibility to heat treatment. The phospholipase A and lipase activities were recovered in a single band when partially purified preparations were subjected to SDS gel electrophoresis. Phospholipase, lysophospholipase, and lipase all required Ca2+ for activity. Phosphatidylcholine, phosphatidylethanolamine, and their lyso analogues were all hydrolysed at equivalent rates and these were substantially greater than the rate of methylpalmitate or tripalmitoylglycerol hydrolyses under similar incubation conditions. Evidence for a direct but slow hydrolysis of the ester at position 2 of phosphoglyceride was obtained; however, release of fatty acid from this position is mostly indirect involving acyl migration to position 1 and subsequent release of the translocated fatty acid. Escherichia coli, therefore, appears to possess a lipolytic enzyme of broad substrate specificity acting mainly at position 1 but also at position 2 of phosphoglycerides and on triacylglycerols and methyl fatty-acid esters.     

Characterization of the transacylase activity of rat liver 60-kDa lysophospholipase-transacylase. Acyl transfer from the sn-2 to the sn-1 position.

Rat liver 60-kDa lysophospholipase-transacylase catalyzes not only the hydrolysis of 1-acyl-sn-glycero-3-phosphocholine, but also the transfer of its acyl chain to a second molecule of 1-acyl-sn-glycero-3-phosphocholine to form phosphatidylcholine (H. Sugimoto, S. Yamashita, J. Biol. Chem. 269 (1994) 6252-6258). Here we report the detailed characterization of the transacylase activity of the enzyme. The enzyme mediated three types of acyl transfer between donor and acceptor lipids, transferring acyl residues from: (1) the sn-1 to -1(3); (2) sn-1 to -2; and (3) sn-2 to -1 positions. In the sn-1 to -1(3) transfer, the sn-1 acyl residue of 1-acyl-sn-glycero-3-phosphocholine was transferred to the sn-1(3) positions of glycerol and 2-acyl-sn-glycerol, producing 1(3)-acyl-sn-glycerol and 1,2-diacyl-sn-glycerol, respectively. In the sn-1 to -2 transfer, the sn-1 acyl residue of 1-acyl-sn-glycero-3-phosphocholine was transferred to not only the sn-2 positions of 1-acyl-sn-glycero-3-phosphocholine, but also 1-acyl-sn-glycero-3-phosphoethanolamine, producing phosphatidylcholine and phosphatidylethanolamine, respectively. 1-Acyl-sn-glycero-3-phospho-myo-inositol and 1-acyl-sn-glycero-3-phosphoserine were much less effectively transacylated by the enzyme. In the sn-2 to -1 transfer, the sn-2 acyl residue of 2-acyl-sn-glycero-3-phosphocholine was transferred to the sn-1 position of 2-acyl-sn-glycero-3-phosphocholine and 2-acyl-sn-glycero-3-phosphoethanolamine, producing phosphatidylcholine and phosphatidylethanolamine, respectively. Consistently, the enzyme hydrolyzed the sn-2 acyl residue from 2-acyl-sn-glycero-3-phosphocholine. By the sn-2 to -1 transfer activity, arachidonic acid was transferred from the sn-2 position of donor lipids to the sn-1 position of acceptor lipids, thus producing 1-arachidonoyl phosphatidylcholine. When 2-arachidonoyl-sn-glycero-3-phosphocholine was used as the sole substrate, diarachidonoyl phosphatidylcholine was synthesized at a rate of 0.23 micromol/min/mg protein. Thus, 60-kDa lysophospholipase-transacylase may play a role in the synthesis of 1-arachidonoyl phosphatidylcholine needed for important cell functions, such as anandamide synthesis.     

Purification and regiospecificity of multiple enzyme activities of phospholipase A(1) from bonito muscle

Phospholipase A(1) (PLA(1)), which catalyzes the hydrolysis of the sn-1 ester bond of diacyl phospholipids, was purified from 100,000 x g supernatant of bonito muscle to homogeneity by ammonium-sulfate precipitation and four consecutive column chromatographies (DEAE anion-exchange, ether-Toyopeal, hydroxylapatite and Toyopeal HW 50S columns). The final preparation showed a single band above the 67-kDa molecular marker on SDS-PAGE, and the molecular mass was determined to be 71.5 kDa by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using bovine serum albumin as a standard for calibration. The N-terminal 8 amino residues were determined to be Ala-Pro-Ala-Glu-Lys-Val-Lys-Try. Regiospecificity of multiple enzyme activities of the PLA(1) was examined using positionally defined synthetic phosphatidylcholine (PC) and lysophosphatidylcholines (LPC). An acyl ester bond at the sn-1 position of PC was exclusively hydrolyzed by phospholipase activity, and 1-acyl LPC was cleaved to fatty acid and glycerophosphocholine by lysophospholipase (LPL) activity. However, the positional isomer, 2-acyl LPC was a poor substrate for LPL activity. PC/transacylation activity was also observed when excess 2-acyl LPC was supplied in the reaction mixture, and fatty acid at the sn-1 position of donor PC was transferred to the sn-1 position of acceptor LPC. These results demonstrate that the multiple enzyme activities of PLA(1), this is lysophospholipase, transacylase as well as phospholipase, have a strict regiospecificity at the sn-1 position of substrates.     

Fatty acyl chain specificity of phosphatidylcholine hydrolysis catalyzed by lipoprotein lipase. Effect of apolipoprotein C-II and its (56-79) synthetic fragment

Mixed acyl chain phosphatidylcholine molecules in Triton N-101 micelles were employed as substrates for lipoprotein lipase to test which substrate acyl chain has the greatest effect on activation of the enzyme by apolipoprotein C-II. The phospholipase A1 activity of lipoprotein lipase was measured by pH-stat. The activation factor (lipoprotein lipase activity plus apolipoprotein C-II/activity minus apolipoprotein C-II) increased monotonically with apolipoprotein C-II concentration up to 1 microM apolipoprotein C-II at an enzyme concentration of 0.01 microM. The maximal activation factor for phosphatidylcholine substrate molecules with sn-2 acyl chain lengths of 14 averages 14.8. By contrast, for sn-2 acyl chain lengths of 16 the activation factor was 29.2. Varying the sn-1 acyl chain length had no significant effect on the activation factor. The chain-length dependence of the activation factor is similar with the apolipoprotein C-II peptide fragment comprising residues 56-79, which does not include the lipid-binding region of apolipoprotein C-II. These data are consistent with a model for activation of lipoprotein lipase in which residues 56-79 bind to lipoprotein lipase and alter the interaction of the sn-2 acyl chain of the phosphatidylcholine (PC) substrate or the lysoPC product within the activated state complex.     

Membrane-bound plasma platelet activating factor acetylhydrolase acts on substrate in the aqueous phase.

Human plasma platelet activating factor acetylhydrolase (pPAF-AH) is a phospholipase A(2) that specifically hydrolyzes the sn-2 ester of platelet activating factor (PAF) and of phospholipids with oxidatively truncated sn-2 fatty acyl chains. pPAF-AH is bound to lipoproteins in vivo, and it binds essentially irreversibly to anionic and zwitterionic phospholipid vesicles in vitro and hydrolyzes PAF and PAF analogues. Substrate hydrolysis also occurs in the absence of vesicles, with a maximum rate reached at the critical micelle concentration. A novel pre-steady-state kinetic analysis with enzyme tightly bound to vesicles and with a substrate that undergoes slow intervesicle exchange establishes that pPAF-AH accesses its substrate from the aqueous phase and thus is not an interfacial enzyme. Such a mechanism readily explains why this enzyme displays dramatic specificity for phospholipids with short sn-2 chains or with medium-length, oxidatively truncated sn-2 chains since a common feature of these lipids is their relatively high water solubility. It also explains why the enzymatic rate drops as the length of the sn-1 chain is increased. pPAF-AH shows broad specificity toward phospholipids with different polar headgroups. Additional results are that PAF undergoes intervesicle exchange on the subminute time scale and it does not undergo transbilayer movement over tens of minutes.     

Hydrolysis of 2-acyl-sn-glycero-3-phosphocholines in guinea pig heart mitochondria

Although both 2-acyl-sn-glycero-3-phosphocholine and 1-acyl-sn-glycero-3-phosphocholine may be produced from phosphatidylcholine hydrolysis, studies on the former have lagged behind that of the latter. In this study a lysophospholipase A2 that hydrolyses 2-acyl-sn-glycero-3-phosphocholine has been characterized in guinea pig heart mitochondria. The lysophospholipase A2 activity was not dependent on Ca2+ and was inhibited differentially by saturated and unsaturated fatty acids. This lysophospholipase A2 activity was able to discriminate among different molecular species of 2-acyl-sn-glycero-3-phosphocholines when they were presented individually or in pairs. The order of decreasing rates of hydrolysis of different molecular species of 2-lysophosphatidylcholines, when the substrates were presented singly, was 18:2 greater than 20:4 greater than 18:1 greater than 16:0. A differential inhibition of the rate of hydrolysis of the individual substrates was observed when the substrates were presented in pairs. The degree of inhibition was dependent on the molar ratio of the mixed substrates. The characteristics of the enzyme suggest that involvement in the selective release of fatty acids from mitochondrial phosphatidylcholine would depend on a high selectivity of phospholipase A1 for different molecular species of phosphatidylcholine. A lysophospholipase A1 activity was also characterized in the mitochondria with a distinct acyl specificity from the lysophospholipase A2. Other characteristics of the two lysophospholipases suggest that the two reactions are not catalysed by the same enzyme.     

Hydrolysis of novel diacylglycerol analogs and phorbol diesters by serum lipase

Rat serum, active in the hydrolysis of the tumor-promoting phorbol diester, 12-O-tetradecanoylphorbol-13-acetate (TPA), was examined with regard to lipid interferences of [3H]TPA hydrolysis and enzyme substrate specificity. The enzymatic hydrolysis of TPA could be enhanced 8-fold, over crude serum, by using a lipid-free acetone powder of rat serum. Addition of lipid to the lipid-free acetone powder produced potent inhibition of TPA hydrolysis. The inclusion of multilamallar liposomes resulted in similar inhibition, and isolation of liposomes by high-speed centrifugation showed that 95% of the radiolabeled TPA was associated with the fatty pellet. Substrate specificity studies demonstrated that the serum activity hydrolyzes the long-chain ester of TPA and the long-chain primary acyl group of diacylglycerols. TPA was hydrolyzed at approximately twice the rate of dioleoylglycerol; however, the most reactive substrates were those synthetic analogs of diacylglycerol containing a short-chain ester group at the sn-2 position. Palmitic acid was liberated from [1-14C]palmitoyl-2-acetyl-sn-glycerol and [1-14C]palmitoyl-2-butyryl-sn-glycerol at 120- and 33-times the rate of TPA hydrolysis, respectively. Lipase resistant 1-hexadecyl-2-[3H]acetylglycerol was also used as substrate, but the sn-2 ester moiety showed poor lability. The diacylglycerol analogs are new lipase substrates and, in view of their similarities to the fatty acyl portion of TPA, it is thought that these compounds could serve as protein kinase C activators.     

Positional specificity of lysosomal phospholipase A2

Lysosomal phospholipase A(2) (Lpla2) is highly expressed in alveolar macrophages and may mediate the phospholipid metabolism of surfactant. Studies on the properties of this phospholipase are consistent with the presence of both phospholipase A(1) and phospholipase A(2) activities. These activities were studied through the production of O-acyl compounds, produced by the transacylase activity of Lpla2. Liposomes containing POPC and N-acetylsphingosine (NAS) were incubated with the soluble fraction obtained from MDCK cells stably transfected with the mouse Lpla2 gene. Two 1-O-acyl-NASs, 1-O-palmitoyl-NAS and 1-O-oleoyl-NAS, were produced by Lpla2. The formation rate of 1-O-oleoyl-NAS was 2.5-fold that of 1-O-palmitoyl-NAS. When 1-oleoyl-2-palmitoyl-sn-glycero-3-phosphocholine (OPPC) was used, the formation rate of 1-O-oleoyl-NAS was 5-fold higher than that of 1-O-palmitoyl-NAS. Thus, Lpla2 can act on acyl groups at both sn-1 and sn-2 positions of POPC and OPPC. When 1-palmitoyl-2-unsaturated acyl-sn-glycero-3-phosphocholines were used as acyl donors, the transacylation of the acyl group from the sn-2 position to NAS was preferred to that of the palmitoyl group from the sn-1 position. An exception was observed for 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC), for which the formation rate of 1-O-palmitoyl-NAS from PAPC was 4-fold greater than that of 1-O-arachidonoyl-NAS. Thus, Lpla2 has broad positional specificity for the sn-1 and sn-2 acyl groups in phosphatidylcholine and phosphatidylethanolamine.     

Conversion of Bacillus thermocatenulatus lipase into an efficient phospholipase with increased activity towards long-chain fatty acyl substrates by directed evolution and rational design.

The thermoalkalophilic lipase from Bacillus thermocatenulatus BTL2 exhibits a low phospholipase activity (lecithin/tributyrin ratio 0.03). A single round of random mutagenesis of the BTL2 gene followed by screening of 6000 transformants on egg-yolk plates identified three variants with 10-12-fold increased phospholipase activities, corresponding to lecithin/tributyrin ratios of 0.16-0.36. All variants were specific for the sn-1 acyl ester bond of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine. Mutations occurred predominantly in the N-terminal part of BTL2 with regions surrounding the predicted helix alpha(4) and lid as hotspots. Two mutations, L184P located in the predicted helix alpha(4) and H15P found in the highly conserved oxy-anion hole motif among hydrolases, were identified to account for increased phospholipase activity. Two of the three variants showed reduced activities towards medium- and long-chain fatty acyl methyl esters compared to the wild-type enzyme. Substitution of Leu353 with Ser, which is located adjacent to the active site histidine and is important for phospholipase activity in the Staphylococcus hyicus lipase, increased the absolute phospholipase activities of the variants, but not of BTL2, approximately 2-fold. The engineered best variant displayed a lecithin/tributyrin ratio of 0.52, corresponding to a 17-fold increase compared to the wild-type enzyme. Moreover, this variant exhibited a 1.5-4-fold higher activity towards long-chain fatty acyl methyl ester (C18:1, C18:2, C18 and C20) compared to BTL2. A second round of mutagenesis and screening on lecithin-plates yielded no new variants with further increased phospholipase/lipase activity ratios, but instead one variant with a 5-fold increased expression rate and two variants with a 3-fold reduced activity towards triolein were obtained.     

Stereochemical and positional specificity of the lipase/acyltransferase produced by Aeromonas hydrophila.

Aeromonas species secrete a glycerophospholipid-cholesterol acyltransferase (GCAT) which shares many properties with mammalian plasma lecithin-cholesterol acetyltransferase (LCAT). We have studied the stereochemical and positional specificity of GCAT against a variety of lipid substrates using NMR spectroscopy as well as other assay methods. The results show that both the primary and secondary acyl ester bonds of L-phosphatidylcholine can be hydrolyzed but only the sn-2 fatty acid can be transferred to cholesterol. The enzyme has an absolute requirement for the L configuration at the sn-2 position of phosphatidylcholine. The secondary ester bond of D-phosphatidylcholine cannot be hydrolyzed, and this lipid is not a substrate for acyl transfer. In contrast to the phospholipases, but similar to LCAT, the enzyme does not interact stereochemically with the phosphorus of phosphatidylcholine. In fact, the phosphorus is not required for enzyme activity, as GCAT will also hydrolyze monolayers of diglyceride, although at much lower rates.     

Synthetic phospholipids as substrates for phospholipase C from Bacillus cereus.

The substrate requirement of phospholipids for hydrolysis with phospholipase C from Bacillus cereus was studied with synthetic lipids well-defined in structure and configuration. For optimal activity, the glycerol molecule must contain three substituents: phosphocholine in sn-3-, an ester bond in sn-2- and an ether- or ester bond in sn-1-position. The length of the ester or ether chains is of minor importance. Any deviation from these structural requirements results in a large decrease in the hydrolysis rate. These essential structural and configurational elements for optimal activity for the B. cereus enzyme are perfectly combined in the platelet activating factor, 1-O-hexadecyl-2-acetyl-sn-glycero-3- phosphocholine. This molecule is one of the best substrates for hydrolysis with the bacterial phospholipase C.     

Substrate specificity of Staphylococcus aureus (TEN5) lipases with isomeric oleoyl-sn-glycerol ethers as substrates

For the first time fully protected substrates with only one hydrolyzable ester bond have been used to analyze the substrate specificity of microbial lipases. In these substrates the ester is attached to the glycerol molecule in a precisely defined position. The use of three different substituents generates chirality and thus allows the analysis of positional specificities of individual lipases. Therefore, these new substrates have been used to study the enzymatic activities of two closely related lipases isolated from Staphylococcus aureus (TEN5) designated the 44 and 43 kDa lipase. The lipases, especially the 44 kDa molecule, show a high specificity for the hydrolysis of the ester in the sn-1 position (S-configuration), which is hydrolyzed by a factor of ten faster than that in the sn-3 position. In addition, the study demonstrates for the first time that the rate of hydrolysis of a fatty acid ester attached to the sn-2 position of glycerol by microbial lipases depends on the configuration of the substrate molecule.     

Platelet-activating factor acetylhydrolases: broad substrate specificity and lipoprotein binding does not modulate the catalytic properties of the plasma enzyme

Platelet-activating factor acetylhydrolases (PAF-AHs) are a group of enzymes that hydrolyze the sn-2 acetyl ester of PAF (phospholipase A(2) activity) but not phospholipids with two long fatty acyl groups. Our previous studies showed that membrane-bound human plasma PAF-AH (pPAF-AH) accesses its substrate only from the aqueous phase, which raises the possibility that this enzyme can hydrolyze a variety of lipid esters that are partially soluble in the aqueous phase. Here we show that pPAF-AH has broad substrate specificity in that it hydrolyzes short-chain diacylglycerols, triacylglycerols, and acetylated alkanols, and displays phospholipase A(1) activity. On the basis of all of the substrate specificity results, it appears that the minimal structural requirement for a good pPAF-AH substrate is the portion of a glyceride derivative that includes an sn-2 ester and a reasonably hydrophobic chain in the position occupied by the sn-1 chain. In vivo, pPAF-AH is bound to high and low density lipoproteins, and we show that the apparent maximal velocity for this enzyme is not influenced by lipoprotein binding and that the enzyme hydrolyzes tributyroylglycerol as well as the recombinant pPAF-AH does. Broad substrate specificity is also observed for the structurally homologous PAF-AH which occurs intracellularly [PAF-AH(II)] as well as for the PAF-AH from the lower eukaryote Physarum polycephalum although pPAF-AH and PAF-AH(II) tolerate the removal of the sn-3 headgroup better than the PAF-AH from P. polycephalum does. In contrast, the intracellular PAF-AH found in mammalian brain [PAF-AH(Ib) alpha 1/alpha 1 and alpha 2/alpha 2 homodimers] is more selectively operative on compounds with a short acetyl chain although this enzyme also displays significant phospholipase A(1) activity.     

Characterization of heparin low-affinity phospholipase A1 present in brain and testicular tissue.

We identified a unique phospholipase A (PLA) with relatively low heparin affinity, which was distinguishable from the heparin-binding secretory PLA2s, in rat, mouse, and bovine brains and testes. The partially purified enzyme was Ca2+-independent at neutral pH but Ca2+-dependent at alkaline pH. It predominantly hydrolyzed phosphatidic acid (PA) in the presence of Triton X-100 and phosphatidylethanolamine (PE) in its absence. When rat brain-derived endogenous phospholipids were used as a substrate, the enzyme released saturated fatty acids in marked preference to unsaturated ones. Consistent with this observation, the enzyme hydrolyzed sn-1 ester bonds in the substrates about 2,000 times more efficiently than sn-2 ones, thereby acting like PLA1. The enzyme also exhibited weak but significant sn-1 lysophospholipase activity. On the basis of its limited tissue distribution, substrate head group specificity and immunochemical properties, this enzyme appears to be identical to the recently cloned PA-preferring PLA1.     

Purification and substrate specificity of Staphylococcus hyicus lipase

The Staphylococcus hyicus lipase gene has been cloned and expressed in Staphylococcus carnosus. From the latter organism the enzyme was secreted into the medium as a protein with an apparent molecular mass of 86 kDa. This protein was purified, and the amino-terminal sequence showed that the primary gene product was indeed cleaved at the proposed signal peptide cleavage site. The protein was purified from large-scale preparations after tryptic digestion. This limited proteolysis reduced the molecular mass to 46 kDa and increased the specific activity about 3-fold. Although the enzyme had a low specific activity in the absence of divalent cations, the activity increased about 40-fold in the presence of Sr2+ or Ca2+ ions. The purified lipase has a broad substrate specificity. The acyl chains were removed from the primary and secondary positions of natural neutral glycerides and from a variety of synthetic glyceride analogues. Thus triglycerides were fully hydrolyzed to free fatty acid and glycerol. The enzyme hydrolyzed naturally occurring phosphatidylcholines, their synthetic short-chain analogues, and lysophospholipids to free fatty acids and water-soluble products. The enzyme had a 2-fold higher activity on micelles of short-chain D-lecithins than on micelles composed of the L-isomers. Thus the enzyme from S. hyicus has lipase activity and also high phospholipase A and lysophospholipase activity.     

Substrate specificity and partial characterization of the PAF-acylhydrolase in human serum that rapidly inactivates platelet-activating factor

The structure of the potent inflammatory mediator, platelet-activating factor, is 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC, PAF-acether). Human sera contain an acid labile factor (ALF) that is a Ca+2-independent 2-acylhydrolase-specific for AGEPC and AGEPC-like molecules. The enzyme functions by catalytically removing the sn-2 acetyl moiety from AGEPC, producing the biologically inactive sn-2 hydroxy form or 2-lyso-GEPC. Incubation of ALF with sn-2 acyl PAF analogs indicated that the enzyme hydrolyzes the sn-2 fatty acid only if the chain length is five carbons or less, the sn-1 position fatty acid length is greater than 10 carbon units, and at least one methyl group is present on the terminal amine of the choline group. The enzyme was active with either an ether or ester linkage at the sn-1 position. ALF is inactivated by heating to 65 degrees C for 30 min. It is pronase and trypsin sensitive but resistant to papain and papain with dithiothreitol. Further characteristics of human ALF indicated a broad pH range of activity with an optimum of pH 6.2 and an isoelectric point of 6.2 to 6.7. The specificity and Ca+2 independence of human ALF sets it apart from phospholipase A2. It is proposed that human ALF be called human serum PAF-acylhydrolase to distinguish it from other hydrolases currently known to exist.     

Evidence for the involvement of tyrosine-69 in the control of stereospecificity of porcine pancreatic phospholipase A2

We have studied the role of Tyr-69 of porcine pancreatic phospholipase A2 in catalysis and substrate binding, using site-directed mutagenesis. A mutant was constructed containing Phe at position 69. Kinetic characterization revealed that the Phe-69 mutant has retained enzymatic activity on monomeric and micellar substrates, and that the mutation has only minor effects on kcat and Km. This shows that Tyr-69 plays no role in the true catalytic events during substrate hydrolysis. In contrast, the mutation has a profound influence on the stereospecificity of the enzyme. Whereas the wild-type phospholipase A2 is only able to catalyse the degradation of sn-3 phospholipids, the Phe-69 mutant hydrolyses both the sn-3 isomers and, at a low (1-2%) rate, the sn-1 isomers. Despite the fact that the stereospecificity of the mutant phospholipase has been altered, Phe-69 phospholipase still requires Ca2+ ions as a cofactor and also retains its specificity for the sn-2 ester bond. Our data suggest that in porcine pancreatic phospholipase A2 the hydroxyl group of Tyr-69 serves to fix and orient the phosphate group of phospholipid monomers by hydrogen bonding. Because no such interaction can occur between the Phe-69 side-chain and the phosphate moiety of the substrate monomer, the mutant enzyme loses part of its stereospecificity but not its positional specificity.