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.     

Aromatic Residues in the Substrate Cleft of RPE65 Protein Govern Retinol Isomerization and Modulate Its Progression

Previously, we showed that mutating RPE65 residue Phe-103 preferentially produces 13-cis-retinol instead of 11-cis-retinol, supporting a carbocation/radical cation mechanism of retinol isomerization. We asked whether this modulation of specificity can occur with residues other than Phe-103 and what role it plays in substrate binding and isomerization. We modeled the substrate-binding cleft of RPE65 to identify residues lining its surface. Many are phenylalanines and tyrosines, including three Phe residues (Phe-61, Phe-312, and Phe-526) forming an arch-like arrangement astride the cleft and Tyr-338. Also, Phe-418 sits at the neck of the cleft, lending a bend to the volume enclosed by the cleft. All mutations of Phe-61, Phe-312, and Phe-418 result in severely impaired or inactive enzyme. However, mutation of Phe-526 and Tyr-338, like Phe-103, decreases 11-cis-retinol formation, whereas increasing the 13-cis isomer. Significantly, 2 of these 3 residues, Phe-103 and Tyr-338, are located on putatively mobile interstrand loops. We propose that residual densities located in the binding cleft of the RPE65 structure represents a post-cleavage snapshot consistent not only with a fatty acid product, as originally modeled, but also an 11-cis-retinol product. Substrate docking simulations permit 11-cis- or 13-cis-retinyl ester binding in this relatively closed cleft, with the latter favored in F103L, F526A, and Y338A mutant structures, but prohibit binding of all-trans-retinyl ester, suggesting that isomerization occurs early in the temporal sequence, with O-alkyl ester cleavage occurring later. These findings provide insight into the mechanism of isomerization central to the visual cycle.     

Kinetic characterization of Escherichia coli outer membrane phospholipase A using mixed detergent-lipid micelles

The substrate specificity of Escherichia coli outer membrane phospholipase A was analyzed in mixed micelles of lipid with deoxycholate or Triton X-100. Diglycerides, monoglycerides, and Tweens 40 and 85 in Triton X-100 are hydrolyzed at rates comparable to those of phospholipids and lysophospholipids. p-Nitrophenyl esters of fatty acids with different chain lengths and triglycerides are not hydrolyzed. The minimal substrate characteristics consist of a long acyl chain esterified to a more or less hydrophilic headgroup as is the case for the substrate monopalmitoylglycol. Binding occurs via the hydrocarbon chain of the substrate; diacyl compounds are bound three to five times better than monoacyl compounds. When acting on lecithins, phospholipase A1 activity is six times higher than phospholipase A2 activity or 1-acyl lysophospholipase activity. Activity on the 2-acyl lyso compound is about two times less than that on the 1-acyl lysophospholipid. The enzyme therefore has a clear preference for the primary ester bond of phospholipids. In contrast to phospholipase A1 activity, phospholipase A2 activity is stereospecific. Only the L isomer of a lecithin analogue in which the primary acyl chain was replaced by an alkyl ether group is hydrolyzed. The D isomer of this analogue is a competitive inhibitor, bound with the same affinity as the L isomer. On these ether analogues the enzyme shows the same preference for the primary acyl chain as with the natural diester phospholipids. Despite its broad specificity, the enzyme will initially act as a phospholipase A1 in the E. coli envelope where it is embedded in phospholipids.     

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.     

Hydrolysis of thioester analogs by rat liver phospholipase A1

The hydrolysis of thioester containing phospholipids by rat liver plasmalemma phospholipase A1 was measured in a continuous spectrophotometric assay. In this assay thioester substrates were employed which, upon hydrolysis, liberated a free thiol which was reacted with 4,4'-dithiopyridine to yield the product 4-thiopyridone that absorbs at 324 nm. Thioester substrates, prepared by chemical synthesis, were used in phospholipid and Triton X-100 micelles for kinetic analysis carried out according to the method of Hendrickson and Dennis (Hendrickson, H.S., and Dennis, E.A. (1984) J. Biol. Chem. 259, 5734-5739). Vmax, Ks, and Km values obtained for various isomers and racemic mixtures of the synthetic thioester analogs are compared with corresponding oxyester substrates. Unnatural sn-1 isomers competitively inhibited the hydrolysis of natural sn-3 isomers of phosphatidylethanolamine and phosphatidic acid. Furthermore, the sn-1 isomer of phosphatidic acid was hydrolyzed by phospholipase A1, but with lower catalytic efficiency than the sn-3 isomer. The presence of a thioester at the sn-1 position did not change the Vmax significantly, as compared to the oxyester phospholipids. When two thioesters were present on the phospholipid molecule, the Vmax was decreased significantly. A convenient synthesis of 1-monothioester analogs of phospholipids is reported. The results presented show the usefulness of the spectrophotometric assay for measuring phospholipase A1 activity as well as the influence of racemic mixtures and thioesters on the hydrolytic rate.     

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.     

The role of aspartic acid-49 in the active site of phospholipase A2. A site-specific mutagenesis study of porcine pancreatic phospholipase A2 and the rationale of the enzymatic activity of [lysine49]phospholipase A2 from Agkistrodon piscivorus piscivorus' venom

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]phospholipase 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.     

Effect of phospholipids on membrane-bound and solubilized mannosyltransferase activity from aortic wall

Phospholipids interact on the membrane-bound and solubilized mannosyltransferase activity. The biosynthesis of Dol-P-Man is strongly inhibited by phosphatidic acid and lysophosphatidylcholine. The effect of phospholipids is not related to stereospecificity. Chemical properties of phospholipids (ester or ether bond, length of fatty acids and polarity of head groups) are not an essential factor for inhibition. The different parameters involved in enzymatic reaction of glycosylation are not modified by phospholipids, in particular the integrity of GDP-[14C]mannose. The inhibitory effect of lysophosphatidylcholine and phosphatidic acid on mannosyltransferase activities is related to their possible formation of micellar structures which definitely induce a conformation change of this enzyme.     

Phospholipase A1 acting on phosphatidic acid in porcine platelet membranes

1-[14C]Palmitoyl-2-[3H]arachidonoyl-sn-glycerol 3-phosphate was hydrolyzed to form [14C]palmitic acid and 2-[3H]arachidonoyl-glycerophosphate by porcine platelet membranes. This phospholipase A1 activity was relatively specific for phosphatidic acid; the addition of several other phospholipids in equimolar amounts did not have a significant effect on the hydrolysis of radiolabeled phosphatidic acid, and the specific activity for phosphatidic acid hydrolysis was 20-fold higher than that of the hydrolysis of phosphatidylcholine, phosphatidylethanolamine, or phosphatidylinositol under the conditions used. This phospholipase A1 acting on phosphatidic acid has properties different from those reported for other phospholipases and lipases present in platelets.     

Modulation of solubilized brain fucosyltransferase activity by phospholipids

Phospholipids interact on Triton X-100 solubilized GDP-fucose: asialofetuin fucosyltransferase (EC 2.4.1.68) isolated from sheep brain. This enzymatic activity is modulated by charged phospholipids. In particular, phosphatidic acid and analogues markedly inhibit the transfer of fucose from GDP-[14C]fucose. Kinetic studies show that phosphatidic acid interacts as a mixed inhibitor: the velocity and affinity of fucosyltransferase for the GDP-fucose and asialofetuin substrates are strongly decreased. However, this inhibitory effect is not related to stereospecificity, and the different parameters involved in the enzymatic reaction of glycosylation are not modified. The nature of fatty acids and chemical bond (ester or ether) occurring in the carbohydrate chain does not modify the behaviour of phosphatidic acid with respect to fucosyltransferase activity. Further, the physical state of phosphatidic acid (gel phase or liquid crystalline phase) has no influence. However, as the inhibition is closely pH-dependent, these data suggest that phosphatidic acid might directly interact with the active site of the enzyme and induce a conformational change.     

Cardiolipin-Specific Phospholipase D Activity in Haemophilus parainfluenzae

A highly active phospholipase D that is specific for cardiolipin was detected in the gram-negative bacterium Haemophilus parainfluenzae. Previously reported phospholipase D preparations have come exclusively from higher plants. The bacterial enzyme hydrolyzed cardiolipin to phosphatidyl glycerol and phosphatidic acid. During the incubation, phosphatidic acid disappeared. Phosphatidyl ethanolamine, methylated phosphatidyl ethanolamines, phosphatidyl choline, and phosphatidyl glycerol were not hydrolyzed when cardiolipin was rapidly hydrolyzed.     

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.     

蜡状芽孢杆菌磷脂酶D的活性形式及HKD活性区域的分子改造对其活性的影响

磷脂酶D是一类特殊的酯键水解酶,它能水解磷脂生成磷脂酸和羟基化合物,并能催化某些含羟基的化合物结合到磷脂的酰基上,形成新的磷脂,在食品和医药领域应用潜力巨大。本研究实现了蜡状芽孢杆菌磷脂酶D的克隆,并在大肠杆菌中成功表达。通常情况下,磷脂酶D存在2个HKD保守序列,以单体形式产生活性;少数原核生物中磷脂酶D只有1个HKD保守序列,以二聚体形式产生活性。通过酵母双杂实验发现,源于蜡状芽孢杆菌的磷脂酶D活性存在形式是单体结构,但其只具有1个HKD保守序列,靠近N端存在1个HRD序列,即HKD中K被R取代。将HRD定点突变为HKD,恢复为经典的2个HKD保守序列,其酶活性提高了10% 左右,蛋白质水平的表达量和稳定性无显著变化。通过定点突变提高磷脂酶D活性,为工业化高效生产新型磷脂奠定了理论基础。     

Anionic phospholipids stimulate an arachidonoyl-hydrolyzing phospholipase A2 from macrophages and reduce the calcium requirement for activity

Mechanisms involved in regulating the activity of intracellular phospholipase A2 enzymes that function in eicosanoid and platelet-activating factor production are poorly understood. The properties of the substrate in the membrane may play a role in modulating phospholipase A2 activity. In this study, the effect of anionic phospholipids, diacylglycerol (DAG) and phosphatidylethanolamine (PE) on the activity of a partially purified, intracellular, arachidonoyl-hydrolyzing phospholipase A2 from the macrophage cell line, RAW 264.7 was studied. For these experiments phospholipase A2 activity was assayed in the presence of 1 microM calcium by measuring the hydrolysis of [3H]arachidonic acid from sonicated dispersions of the ether-linked substrate, 1-O-hexadecyl-2[3H]arachidonoylglycerophosphocholine. All the anionic phospholipids tested, including phosphatidylserine (PS), phosphatidic acid (PA), phosphatidylinositol (PI) and phosphatidylinositol-4,5-bisphosphate (PIP2), stimulated phospholipase A2 activity. At the lowest concentration of anionic phospholipids tested. PIP2 was the most stimulatory, resulting in a 7-fold increase in phospholipase A2 activity at 1 mol%. Co-dispersion of either DAG or PE with the substrate also induced a dose-dependent increase in phospholipase A2 activity, whereas sphingomyelin was inhibitory suggesting that the phospholipase A2 more readily hydrolyzed the ether-linked substrate when there was a decrease in the packing density of the bilayer. PIP2, together with either DAG or PE, synergistically stimulated phospholipase A2 activity by about 20-fold, and dramatically decreased the calcium concentration (from mM to nM) required for full activity of the enzyme. The results of this study demonstrate that the presence of anionic phospholipids and the packing characteristics of the bilayer can have pronounced effects on the activity and calcium requirement of an intracellular, arachidonoyl-hydrolyzing phospholipase A2 from macrophages.     

Site-specific mutagenesis of lysine-204, tyrosine-224, tyrosine-228, and histidine-307 of porcine kidney D-amino acid oxidase and the implications as to its catalytic function

In order to evaluate the possible contributions of Lys-204, Tyr-224, Tyr-228, and His-307 in porcine kidney D-amino acid oxidase [EC 1.4.3.3] (DAO) to its catalytic function, we constructed four point mutant cDNAs encoding enzymes possessing Glu-204, Phe-224, Phe-228, and Leu-307 by oligonucleotide-directed in vitro mutagenesis. The four mutant cDNAs and the wild type cDNA could be expressed in vitro with similar efficiencies and about 200 ng of each enzyme protein was produced from 5 micrograms of the respective capped RNA. The electrophoretic mobilities of the in vitro synthesized mutant enzymes on SDS-polyacrylamide gel were almost identical with that of the wild type DAO, and the molecular weight was calculated to be 38,000. The Glu-204 and Phe-224 mutant DAOs showed comparable enzyme activities to that of the wild type enzyme, and were inhibited strongly by sodium benzoate, a potent competitive inhibitor of DAO. The kinetic parameters of the two mutant DAOs were also comparable to those of the wild type DAO. On the other hand, the Phe-228 and Leu-307 mutant DAOs showed no detectable activity. The results indicate that Tyr-228 and His-307 play important roles as to the constitution of the active site or participate in the reaction directly, while Lys-204 and Tyr-224 are not essential in the enzyme reaction.     

Implications of a consensus recognition site for phosphatidylcholine separate from the active site in cobra venom phospholipases A2.

A model structure of Naja naja kaouthia cobra venom phospholipase A2 has been constructed by utilizing molecular modeling techniques. Analysis of the model and available biochemical data reveal the presence in this enzyme of a putative recognition site for choline derivatives in loop 57-70 made up of residues Trp-61, Tyr-63, Phe-64, and Lys-65, together with Glu-55. The magnitude and shape of the electrostatic potential in this binding site are approximately 80% similar to that in the McPC603 antibody binding site specifically recognizing phosphocholine. Docking studies indicate that the recognition site we now describe and the phosphocholine head of an n-alkylphosphocholine molecule are complementary both sterically and electronically, mainly due to anion-cation and cation-pi interactions. Moreover, binding enthalpies of n-heptylphosphocholine to this site are found to parallel the catalytic rate of pancreatic, mutant pancreatic, and cobra venom phospholipase A2 enzymes acting on dihexanoylphosphatidylcholine micelles, suggesting that it behaves as an activator site. This proposal is in keeping with the "dual phospholipid" model put forward to account for the phenomenon of interfacial activation. This novel site is also shown to be able to discriminate choline derivatives from ethanolamine derivatives, in accord with experimental data. On the basis of the results obtained, two functions are assigned to this putative activator site: (i) desolvation of the lipid-enzyme interface, particularly the surroundings of tyrosine at position 69 (Tyr-63), and (ii) opening of the entrance to the active site by means of a conformational change of Tyr-63 whose chi 2 angle rotates nearly 60 degrees.     

The role of Asp-49 and other conserved amino acids in phospholipases A2 and their importance for enzymatic activity

The role of aspartic acid-49 (Asp-49) in the active site of porcine pancreatic phospholipase A2 was studied by recombinant DNA techniques: two mutant proteins were constructed containing either glutamic acid (Glu) or lysine (Lys) at position 49. 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 naturally occurring Lys-49 phospholipase A2 from the venom of Agkistrodon piscivorus piscivorus yielded a protein that was nearly inactive. Inhibition studies showed that this residual activity was due to a small amount of contaminating enzyme and that the Lys-49 homologue itself has no enzymatic activity. Our results indicate that Asp-49 is essential for the catalytic action of phospholipase A2. The importance of Asp-49 was further evaluated by comparison of the primary sequences of 53 phospholipases A2 and phospholipase homologues showing that substitutions at position 49 are accompanied by structural variations of otherwise conserved residues. The occurrence of several nonconserved substitutions appeared to be a general characteristic of nonactive phospholipase A2 homologues.     

The stereoselectivity of the Clostridium perfringens phospholipase C: hydrolysis of thiophosphate analogs of phosphatidylcholine

Thiophosphate containing analogs of phosphatidylcholine have been synthesized with varying degrees of structural complexity. These analogs have been used in a continuous spectrophotometric assay for phospholipase C from Clostridium perfringens in order to examine the requirement for substrate ester functionalities and the stereoselectivity of the enzyme. The substrate analogs with ester groups in the nonpolar portion of the molecule were acceptable substrates for phospholipase C, while those analogs without ester functionalities were not hydrolyzed. Substrate analogs with chiral centers were resolved using the stereospecificity of phospholipase A2 from Crotalus atrox venom. These resolved substrates were used to study the biphasic hydrolytic time courses observed when rac-dioctanoylphosphatidylthiocholine was used as substrate. The "naturally occurring" enantiomer with R absolute configuration was rapidly hydrolyzed in the presence of phospholipase C while the "nonnaturally occurring" enantiomer with S configuration was slowly hydrolyzed only after a long induction or "lag" period. The selectivity for the R enantiomer over the S enantiomer can be lessened by altering the composition of the substrate micelles resulting in accelerated rates of hydrolysis of the S enantiomer.     

Alteration of specific activity and stability of thermostable neutral protease by site-directed mutagenesis.

On the basis of three-dimensional information, many amino acid substitutions were introduced in the thermostable neutral protease (NprM) of Bacillus stearothermophilus MK232 by site-directed mutagenesis. When Glu at position 143 (Glu-143), which is one of the proposed active sites, was substituted for by Gln and Asp, the proteolytic activity disappeared. F114A (Phe-114 to Ala), Y110W (Tyr-110 to Trp), and Y211W (Tyr-211 to Trp) mutant enzymes had higher activity (1.3- to 1.6-fold) than the wild-type enzyme. When an autolysis site, Tyr-93, was replaced by Gly and Ser, the remaining activities of those mutant enzymes were higher than that of the wild-type enzyme.     

Alteration of specific activity and stability of thermostable neutral protease by site-directed mutagenesis.

On the basis of three-dimensional information, many amino acid substitutions were introduced in the thermostable neutral protease (NprM) of Bacillus stearothermophilus MK232 by site-directed mutagenesis. When Glu at position 143 (Glu-143), which is one of the proposed active sites, was substituted for by Gln and Asp, the proteolytic activity disappeared. F114A (Phe-114 to Ala), Y110W (Tyr-110 to Trp), and Y211W (Tyr-211 to Trp) mutant enzymes had higher activity (1.3- to 1.6-fold) than the wild-type enzyme. When an autolysis site, Tyr-93, was replaced by Gly and Ser, the remaining activities of those mutant enzymes were higher than that of the wild-type enzyme.