Substrate specificity for interfacial catalysis by phospholipase A2 in the scooting mode

Action of pig pancreatic phospholipase A2 on vesicles and micelles of homologous anionic phospholipids is examined in the absence of additives. As shown elsewhere (Jain et al. (1986) Biochim. Biophys. Acta 860, 435-447), hydrolysis of anionic vesicles occurs by interfacial catalysis in the scooting mode, i.e., the catalytic turnover is fast relative to the off-rate of the enzyme from the interface. When the rate of intervesicle exchange of the enzyme is negligibly slow, it hydrolyses only the substrate molecules in the outer monolayer of the vesicle to which it is bound. Interfacial catalysis in the scooting mode with a high processivity occurs on vesicles of anionic phospholipids, and under these conditions the dynamics and order of the substrate in the interface influences the catalytic turnover only moderately, i.e., about 2- to 10-fold. Similarly, anomalous kinetic effects of the thermotropic gel-fluid phase transition or of a change in the general disorder of the bilayer organization (fluidity) has a minor effect on the kinetics of hydrolysis in the scooting mode. Similarly, higher unsaturation and shorter acyl chains in the substrate modestly increase the rate of catalytic turnover by the low-calcium form of the enzyme without noticeably influencing the affinity of the enzyme for the interface. On the other hand, perturbation of the charge distribution in the substrate interface can shift the proportion of the bound enzyme by several orders of magnitude. For example, the membrane perturbing amphiphiles (e.g., mepacrine, indomethacin, compound 48/80, aristolochic acid, local anesthetics, and the products of hydrolysis) do not influence the catalytic turnover of the bound enzyme but the proportion of the bound enzyme. Short-chain anionic phospholipids are readily hydrolyzed by phospholipase A2. Now no anomalous increase in the rate of hydrolysis is observed at the critical micelle as is the case with the zwitterionic analogs. This is because with anionic (but not with zwitterionic) substrates the enzyme forms an aggregated complex below the cmc of the monomer. The stability of these micellar complexes does not appear to change noticeably with the acyl chain length of the monomers. These observations show that the factors regulating the quality of interface substantially influence the binding of the enzyme, but not the catalytic turnover in the interface.     

Catalytic cycle of the phosphatidylcholine-preferring phospholipase C from Bacillus cereus. Solvent viscosity, deuterium isotope effects, and proton inventory studies

The phosphatidylcholine-preferring phospholipase C from Bacillus cereus (PLCBc) is a 28.5 kDa enzyme with three zinc ions in its active site. Although much is known about the roles that various PLCBc active site amino acids play in binding and catalysis, there is little information about the rate-determining step of the PLCBc-catalyzed hydrolysis of phospholipids and the catalytic cycle of the enzyme. To gain insight into these aspects of the hydrolysis, solvent viscosity variation experiments were conducted to determine whether an external step (substrate binding or product release) or an internal step (hydrolysis) is rate-limiting. The data indicate that the PLCBc-catalyzed reaction is unaffected by changes in solvent viscosity. This observation is inconsistent with the notion of substrate binding or product release being rate-determining and supports the hypothesis that a chemical step is rate-limiting. Furthermore, a deuterium isotope effect of 1.9 and a linear proton inventory plot indicate one proton is transferred in the rate-determining step. These data may be used to formulate a comprehensive catalytic cycle that is for the first time based on experimental evidence. In this mechanism, Asp55 of PLCBc activates an active site water molecule for attack on the phosphodiester bond, the hydrolysis of which is rate-limiting. The phosphorylcholine product is the first to leave the active site, followed by diacylglycerol.     

Adsorption of pancreatic (pro)phospholipase A2 to various physiological substrates

The adsorption of pancreatic phospholipase was studied in vitro in the presence of egg yolk lipoprotein emulsion, Intralipid emulsion, and milk fat globules. When the emulsions are incubated with bile salts, the latter dissociate a considerable fraction of the phospholipids initially associated with the emulsions, leading to the coexistence of an emulsified phase and a phase of mixed micelles. After the addition of pancreatic phospholipase A2, gel filtration shows that the enzyme was more than 90% bound to mixed micelles, regardless of the type of emulsion used. Comparable results were obtained by replacing the bile salts with human gallbladder bile. In parallel, pancreatic zymogen was never found to be bound to any of the lipid structures present (emulsion or mixed micelles). When the catalytic site of pancreatic phospholipase A2 was blocked with 4-bromophenacylbromide, there was no fixation on mixed micelles. Fixation was restored by the presence of lysolecithins and fatty acids in the incubation mixtures. The partial transformation of all emulsified substrates to mixed micelles by bile salts in vivo would thus lead to optimum activity of pancreatic phospholipase A2.     

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.     

Kinetic characterization of phospholipase A2 modified by manoalogue

Manoalogue, a synthetic analogue of the sea sponge-derived manoalide, has been previously shown to partially inactivate the phospholipase A2 from cobra venom (Reynolds, L. J., Morgan, B. P., Hite, E. D., Mihelich, E. D., & Dennis, E. A. (1988) J. Am. Chem. Soc. 110, 5172) by reacting with enzyme lysine residues. In the present study, the inactivation of the phospholipases A2 from pig pancreas, bee venom, and cobra (Naja naja naja) venom by manoalogue was studied in detail. Manoalogue-treated enzymes were examined in the scooting mode on vesicles of 1,2-dimyristoyl-sn-glycero-3-phosphomethanol. Here the native enzymes bound irreversibly to the vesicles and hydrolyzed all of the phospholipids in the outer monolayer without leaving the surface of the interface. All three manoalogue-treated enzymes showed reduced catalytic turnover for substrate hydrolysis in the scooting mode, and the modified enzymes did not hop from one vesicle to another. Thus, inactivation by manoalogue is not due to the decrease in the fraction of enzyme bound to the substrate interface. This result was also confirmed by fluorescence studies that directly monitored the binding of phospholipase A2 to vesicles. A chemically modified form of the pig pancreatic phospholipase A2 in which all of the lysine epsilon-amino groups have been amidinated was not inactivated by manoalogue, indicating that the modification of lysine residues and not the amino-terminus is required for the inactivation. Several studies indicated that the manoalogue-modified enzymes contain a functional active site. For example, studies that monitored the protection by ligands of the active site from attack by a alkylating agent showed that manoalogue-modified pig phospholipase A2 was capable of binding calcium, a substrate analogue, lipolysis products, and a competitive inhibitor. Furthermore, relative to native enzymes, manoalogue-modified enzymes retained significantly higher catalytic activities when acting on water-soluble substrates than when acting on vesicles in the scooting mode. Intact manoalogue had no affinity for the catalytic site on the enzyme as it did not inhibit the enzyme in the scooting mode and it did not protect the active site from alkylation. Pig pancreatic phospholipase A2 bound to micelles of 2-hexadecyl-sn-glycero-3-phosphocholine was resistant to inactivation by manoalogue, suggesting that the modification of lysine residues on the interfacial recognition surface of the enzyme was required for inactivation.(ABSTRACT TRUNCATED AT 400 WORDS)     

A continuous fluorescence displacement assay for the measurement of phospholipase A2 and other lipases that release long-chain fatty acids.

1. A new continuous fluorescence assay for phospholipase A2 is described which involves the displacement of the highly fluorescent fatty-acid probe 11-(dansylamino)undecanoic acid from rat liver fatty-acid-binding protein by long-chain fatty acids released as a result of phospholipase A2-catalysed hydrolysis of phospholipids. The initial rate of decrease in fluorescence is linearly related to enzyme activity. 2. The assay will detect enzyme activity down to about 10 pmol/min per ml and gives a linear response up to about 10 nmol/min per ml. 3. The assay will work with all phospholipids that have been tested including phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol and phosphatidylglycerol. Substrates carrying a net negative charge showed the highest rates of hydrolysis. 4. The assay will work, in principle, with an enzyme catalysing the release of long-chain fatty acids from a fatty-acylated substrate. This has been confirmed with pancreatic lipase and cholesterol esterase.     

18O isotope exchange experiments on phospholipase A2 determined by 13C-NMR: monomeric phosphatidylcholine and micellar phosphatidylethanolamine substrates

Experiments were carried out to determine whether the hydrolytic step or the product release step is the rate-limiting step for non-activated phospholipase A2 hydrolysis (Dennis, E.A. (1983) in The Enzymes, 3rd Edn., Vol 16 (Boyer, P., ed.), pp. 307-353, Academic Press, New York) of mixed micelles of phosphatidylethanolamine and Triton X-100 in the absence of activator phospholipids and of monomeric short chain phosphatidylcholine in the absence of an interface (Lombardo, D. et al. (1986) J. Biol. Chem. 261, 11663-11666). Phospholipase A2-catalyzed exchange of H2(18)O into 1-alkyl-2-[1(13)C]lauroyl-sn-glycero-3-phosphorylethanolamine and into 1-hexanoyl-2-[1-13C]hexanoyl-sn-glycero-3-phosphorylcholine were examined. Incorporation of 18O was detected by the effect of 18O on 13C chemical shifts in 13C-NMR. Both the substrate and products of the reactions were examined for 18O incorporation. 18O was incorporated into the fatty acid product, but no incorporation of 18O into the substrate was found. These results suggest that the hydrolytic step is not followed by a higher energy transition state and that it, or a step before it, is rate-limiting. Coupled with kinetic experiments, this strongly suggests that the hydrolytic step is the rate-limiting step. Thus, the role of micellar and membrane interfaces in phospholipase A2 reactions does not appear to be by aiding product removal from the enzyme active site.     

Transformation of Phospholipids by Cabbage Phospholipase D in Mixed Micelles Containing 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate

We compared the activities of cabbage phospholipase D during hydrolysis and transesterification of phosphatidylcholine in mixed micelles of surface-active compounds with various physicochemical properties. Mixed micelles of phospholipids and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (ratio, 1 : 2) were among the best substrates. Hydrolysis and transphosphatidylation were studied in micelles containing 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Mixed micelles of phosphatidylcholine and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate may serve as a new substrate for the measurement of phospholipase D activity and preparation of phospholipids using this enzyme.     

Transformation of phospholipids by cabbage phospholipase D in mixed micelles containing 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate

We compared the activities of cabbage phospholipase D during hydrolysis and transesterification of phosphatidylcholine in mixed micelles of surface-active compounds with various physicochemical properties. Mixed micelles of phospholipids and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (ratio, 1:2) were among the best substrates. Hydrolysis and transphosphatidylation were studied in micelles containing 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Mixed micelles of phosphatidylcholine and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate may serve as a new substrate for measurements of phospholipase D activity and preparative isolation of phospholipids using this enzyme.     

Enzymatic hydrolysis of short-chain lecithin/long-chain phospholipid unilamellar vesicles: sensitivity of phospholipases to matrix phase state

Short-chain lecithin/long-chain phospholipid unilamellar vesicles (SLUVs), unlike pure long-chain lecithin vesicles, are excellent substrates for water-soluble phospholipases. Hemolysis assays show that greater than 99.5% of the short-chain lecithin is partitioned in the bilayer. In these binary component vesicles, the short-chain species is the preferred substrate, while the long-chain phospholipid can be treated as an inhibitor (phospholipase C) or poor substrate (phospholipase A2). For phospholipase C Bacillus cereus, apparent Km and Vmax values show that bilayer-solubilized diheptanoylphosphatidylcholine (diheptanoyl-PC) is nearly as good a substrate as pure micellar diheptanoyl-PC, although the extent of short-chain lecithin hydrolysis depends on the phase state of the long-chain lipid. For phospholipase A2 Naja naja naja, both Km and Vmax values show a greater range: in a gel-state matrix, diheptanoyl-PC is hydrolyzed with micellelike kinetic parameters; in a liquid-crystalline matrix, the short-chain lecithin becomes comparable to the long-chain component. Both enzymes also show an anomalous increase in specific activity toward diheptanoyl-PC around the phase transition temperature of the long-chain phospholipid. Since the short-chain lecithin does not exhibit a phase transition, this must reflect fluctuations in head-group area or vertical motions of the short-chain lecithin caused by surrounding long-chain lecithin molecules. These results are discussed in terms of a specific model for SLUV hydrolysis and a general explanation for the "interfacial activation" observed with water-soluble phospholipases.     

Anchoring of phospholipase A2: the effect of anions and deuterated water, and the role of N-terminus region

The effect of anions and deuterated water on the kinetics of action of pig pancreatic phospholipase A2 is examined to elaborate the role of ionic interactions in binding of the enzyme to the substrate interface. Anions and deuterated water have no significant effect on the hydrolysis of monomeric substrates. Hydrolysis of vesicles of DMPMe (ester) is completely inhibited in deuterated water. The shape of the reaction progress curve is altered in the presence of anions. The nature and magnitude of the effect of anions depends upon the nature of the substrate as well as of the anion. Substantial effects of anions on the reaction progress curve are observed even at concentrations below 0.1 M and the sequence of effectiveness for DMPMe vesicles is sulfate greater than chloride greater than thiocyanate. Apparently, anions in the aqueous phase bind to the enzyme, and thus compete with the anionic interface for binding to the enzyme. Binding of the enzyme to anionic groups on the interface results in activation and increased accessibility of the catalytic site possibly via hydrogen bonding network involving water molecule. In order to elaborate the role of the N-terminus region in interfacial anchoring, the action of several semisynthetic pancreatic phospholipase A2s is examined on vesicles of anionic and zwitterionic phospholipids. The first-order rate constant for the hydrolysis of DMPMe in the scooting mode by the various semisynthetic enzymes is in a narrow range: 0.7 +/- 0.15 per min for phospholipase A2 derived from pig pancreas and 0.8 +/- 0.4 per min for the enzymes derived from bovine pancreas. In all cases a maximum of about 4300 substrate molecules are hydrolyzed by each phospholipase A2 molecule. If anions are added at the end of the first-order reaction progress curve, a pseudo-zero-order reaction progress curve is observed due to an increased intervesicle exchange of the bound enzyme. These rates are found to be considerably different for different enzymes in which one or more amino acids in the N-terminus region have been substituted. Steady-state and fluorescence life-time data for these enzymes in water, 2H2O and in the presence of lipids is also reported. The kinetic and binding results are interpreted to suggest that the N-terminus region of phospholipase A2 along with some other cationic residues are involved in anchoring of phospholipase A2 to the interface, and the catalytically active enzyme in the interface is monomeric.     

Interfacial catalysis by phospholipase A2: substrate specificity in vesicles.

The binding equilibrium of phospholipase A2 (PLA2) to the substrate interface influences many aspects of the overall kinetics of interfacial catalysis by this enzyme. For example, the interpretation of kinetic data on substrate specificity was difficult when there was a significant kinetic contribution from the interfacial binding step to the steady-state catalytic turnover. This problem was commonly encountered with vesicles of zwitterionic phospholipids, where the binding of PLA2 to the interface was relatively poor. The action of PLA2 on phosphatidylcholine (PC) vesicles containing a small amount of anionic phospholipid, such as phosphatidic acid (PA), was studied. It was shown that the hydrolysis of these mixed lipid vesicles occurs in the scooting mode in which the enzyme remains tightly bound to the interface and only the substrate molecules present on the outer monolayer of the target vesicle became hydrolyzed Thus the phenomenon of scooting mode hydrolysis was not restricted to the action of PLA2 on vesicles of pure anionic phospholipids, but it was also observed with vesicles of zwitterionic lipids as long as a critical amount of anionic compound was present. Under such conditions, the initial rate of hydrolysis of PC in the mixed PC/PA vesicles was enhanced more than 50-fold. Binding studies of PLA2 to vesicles and kinetic studies in the scooting mode demonstrated that the enhancement of PC hydrolysis in the PC/PA covesicles was due to the much higher affinity of the enzyme toward covesicles compared to vesicles of pure PC phospholipids. A novel and technically simple protocol for accurate determination of the substrate specificity of PLA2 at the interface was also developed by using a double-radiolabel approach. Here, the action of PLA2 in the scooting mode was studied on vesicles of the anionic phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphomethanol that contained small amounts of 3H- and 14C-labeled phospholipids. From an analysis of the 3H and 14C radioactivity in the released fatty acid products, the ratio of substrate specificity constants (kcat/KMS) was obtained for any pair of radiolabeled substrates. These studies showed that the PLA2s from pig pancreas and Naja naja naja venom did not discriminate between phosphatidylcholine and phosphatidylethanolamine phospholipids or between phospholipids with saturated versus unsaturated acyl chains and that the pig enzyme had a slight preference for anionic phospholipids (2-3-fold). The described protocol provided an accurate measure of the substrate specificity of PLA2 without complications arising from the differences in binding affinities of the enzyme to vesicles composed of pure phospholipids.     

Effect of phospholipase D on micellar phospholipids and the role of calcium ions]

The rates of the reaction products formation under simultaneous phospholipase D effect on phosphatidyl ethanolamine and phosphatidyl choline were studied. The hydrolysis of cephalin, unlike the phospholipase D effect on lecithin, does not require Ca2+ ions. Ca2+ does not affect the enzymatic degradation of lecithin and inhibits the reaction with cephalin in "inorganized" phospholipid emulsions. The hydrolysis of micellar phospholipids by phospholipase D (in the presence of the anionic detergent sodium dodecyl sulfate) is accelerated by Ca2+ ions for both substrates. The apparent Km value is equal to 1.5 mM and does not depend on the phospholipid type. In contrast, the value of kcat for lecithin is twice as high as that for cephalin. It was demonstrated that the phase state of the phospholipids and the chemical nature of the alcohol residue in the phospholipid molecule are essential for the substrate specificity of phospholipase D.     

Heparin prevents the binding of phospholipase A2 to phospholipid micelles: importance of the amino-terminus

The activity of the major isoform of porcine pancreatic phospholipase A2 (PLA2), designated B-PLA2, against micellar substrates is inhibited by heparin. Inhibition is a consequence of binding of the enzyme to heparin, documented by a heparin-induced alteration in the intrinsic fluorescence of B-PLA2 and in the 8-anilino-1-naphthalene sulfonate fluorescence and by the enhanced rate of chemical modification of the active site residue His-48. As a consequence of heparin binding, the conformation of B-PLA2 at the active site and at the amino-terminus is altered, and the enzyme does not bind to phospholipid micelles. In spite of the heparin-induced conformational changes, B-PLA2 retains its ability to catalyze the hydrolysis of monomeric phospholipid. Other glycosaminoglycans can bind to and inhibit the activity of B-PLA2 toward organized phospholipids, but none tested is as effective as heparin. An isoform of the pancreatic enzyme, designated UB-PLA2 and which corresponds to iso-pig PLA2, does not bind to nor is its catalytic activity influenced by heparin. A peptide corresponding to the amino-terminal 26 residues of B-PLA2 can rescue PLA2 from heparin inhibition. A similar peptide corresponding to the amino-terminus of UB-PLA2 has no effect on heparin inhibition. A model for the inhibition of B-PLA2 by heparin is proposed in which the catalytically significant effect of heparin is to interact directly with the amino-terminus of B-PLA2, the interfacial recognition site, to prevent the enzyme from binding to micellar substrates.     

On the substrate specificity of rat liver phospholipase A1

The substrate specificity of purified phospholipase A1 was studied using mixed micelles of phospholipid and Triton X-100. The kinetic analysis employed determined Vmax, Ks (a dissociation constant for the phospholipase A1-mixed micelle complex), and Km (the Michaelis constant for the catalytic step which reflects the binding of the enzyme to the substrate in the interface). The order of Vmax values was phosphatidic acid greater than phosphatidylethanolamine greater than phosphatidylcholine greater than phosphatidylserine. The order of Ks values was phosphatidylcholine greater than phosphatidylethanolamine greater than phosphatidic acid greater than phosphatidylserine; the order of Km values was phosphatidic acid greater than phosphatidylethanolamine = phosphatidylserine greater than phosphatidylcholine. When present together, phosphatidylcholine inhibited the hydrolysis of phosphatidylethanolamine but phosphatidylethanolamine did not affect the hydrolysis of phosphatidylcholine. Sphingomyelin, phosphatidylcholine plasmalogen, and phosphatidylethanolamine plasmalogen had no effect on the hydrolysis of phosphatidylethanolamine. The effects of the reaction products, lysolipids and/or fatty acids, were also considered for their influence on phosphatidylethanolamine hydrolysis catalyzed by phospholipase A1. Free fatty acid was found to inhibit, whereas lysophospholipids stimulated hydrolysis of phosphatidylethanolamine. In a mixture of 1,2- and 1,3-diacylglycerides in mixed micelles, only the acyl chain at the sn-1 position of the 1,2 compound was hydrolyzed. Surface charge did not modulate the hydrolysis of phosphatidylcholine vesicles or mixed micelles. In conclusion, it is hypothesized that steric hindrance at position 3 of the glycerol regulates substrate binding in the active site and that an acyl group in position 1 is favored over a vinyl ether linkage for binding.     

Interfacial catalysis by phospholipase A2: evaluation of the interfacial rate constants by steady-state isotope effect studies.

The kinetics of hydrolysis of phospholipid vesicles by phospholipase A2 (PLA2) in the scooting mode can be described by the Michaelis-Menten formalism for the action of the enzyme in the interface (E*). E* + S in equilibrium E*S in equilibrium E*P in equilibrium E* + Products The values of the interfacial rate constants cannot be obtained by classical methods because the concentration of the substrate within the lipid bilayer is not easily manipulated. In the present study, carbonyl-carbon heavy atom isotope effects for the hydrolysis of phospholipids have been measured in both vesicles and in mixed micelles in which the phospholipid was present in the nonionic detergent Triton X-100. A large [14C]carbonyl carbon isotope effect of 1.12 +/- 0.02 was measured for the cobra venom PLA2-catalyzed hydrolysis of dipalmitoylphosphatidylcholine in Triton X-100. In contrast, no isotope effect (1.01 +/- 0.01) was measured for the action of the porcine pancreatic and cobra venom enzymes on vesicles of dimyristoylphosphatidylmethanol in the scooting mode. In a second experiment, the hydrolysis of vesicles was carried out in oxygen-18 enriched water. Analysis of the released fatty acid product by mass spectrometry showed that it contained only a single oxygen-18. All of these results were used to estimate both the forward and reverse commitments to catalysis. The lack of doubly labeled fatty acid demonstrated that the product is released from the E*P complex faster than the reverse of the esterolysis step. The small isotope effect in vesicles demonstrated that the E*S complex goes on to products faster than substrate is released from the enzyme. The relevance of these results to an understanding of substrate specificity and inhibition of PLA2 is discussed. In addition, the conditions placed on the values of the rate constants obtained in the present study together with results obtained in the other studies described in this series of papers have led to the evaluation of most of the interfacial rate constants for the hydrolysis of phospholipid vesicles by PLA2.     

Rate-determining step of butyrylcholinesterase-catalyzed hydrolysis of benzoylcholine and benzoylthiocholine. Volumetric study of wild-type and D70G mutant behavior.

The rate-limiting step for hydrolysis of the positively charged oxoester benzoylcholine (BzCh) by human butyrylcholinesterase (BuChE) is deacylation (k(3)), whereas it is acylation (k(2)) for hydrolysis of the homologous thioester benzoylthiocholine (BzSCh). Steady-state hydrolysis of BzCh and BzSCh by wild-type BuChE and its peripheral anionic site mutant D70G was investigated at different hydrostatic pressures, which allowed determination of volume changes associated with substrate binding, and the activation volumes for the chemical steps. A differential nonlinear pressure-dependence of the catalytic parameters for hydrolysis of both substrates by both enzymes was shown. Nonlinearity of the plots may be explained in terms of compressibility changes or rate-limiting changes. To distinguish between these two possibilities, enzyme phosphorylation by diisopropylfluorophosphate (DFP) in the presence of substrate (BzSCh) under pressure was studied. There was no pressure dependence of volume changes for DFP binding or for phosphorylation of either wild-type or D70G. Analysis of the pressure dependence for steady-state hydrolysis of substrates, and for phosphorylation by DFP provided evidence that no enzyme compressibility changes occurred during the catalyzed reactions. Thus, the nonlinear pressure dependence of substrate hydrolysis reflects changes in the rate-limiting step with pressure. Change in rate-determining step occurred at a pressure of 100 MPa for hydrolysis of BzCh by wild-type and at 75 MPa for D70G. For hydrolysis of BzSCh the change occurred at higher pressures because k(2) < k(3) at atmospheric pressure for this substrate. Elementary volume change contributions upon initial binding, productive binding, acylation and deacylation were calculated from the pressure differentiation of kinetic constants. This analysis shed light on the molecular events taking place along the hydrolysis pathways of BzCh and BzSCh by wild-type BuChE and the D70G mutant. In addition, volume change differences between wild-type and D70G provided new evidence that residue D70 in the peripheral site controls hydration of the active site gorge and the dynamics of the water molecule network during catalysis. Finally, a steady-state kinetic study of the oxyanion hole mutant (G117H) showed that substitution of the ethereal sulfur for oxygen in the substrate alters the final adjustment of substrate in the active site and stabilization of the acylation transition state.     

Hydrolysis of oxo- and thio-esters by human butyrylcholinesterase

Catalytic parameters of human butyrylcholinesterase (BuChE) for hydrolysis of homologous pairs of oxo-esters and thio-esters were compared. Substrates were positively charged (benzoylcholine versus benzoylthiocholine) and neutral (phenylacetate versus phenylthioacetate). In addition to wild-type BuChE, enzymes containing mutations were used. Single mutants at positions: G117, a key residue in the oxyanion hole, and D70, the main component of the peripheral anionic site were tested. Double mutants containing G117H and mutations on residues of the oxyanion hole (G115, A199), or the pi-cation binding site (W82), or residue E197 that is involved in stabilization of tetrahedral intermediates were also studied. A mathematical analysis was used to compare data for BuChE-catalyzed hydrolysis of various pairs of oxo-esters and thio-esters and to determine the rate-limiting step of catalysis for each substrate. The interest and limitation of this method is discussed. Molecular docking was used to analyze how the mutations could have altered the binding of the oxo-ester or the thio-ester. Results indicate that substitution of the ethereal oxygen for sulfur in substrates may alter the adjustment of substrate in the active site and stabilization of the transition-state for acylation. This affects the k2/k3 ratio and, in turn, controls the rate-limiting step of the hydrolytic reaction. Stabilization of the transition state is modulated both by the alcohol and acyl moieties of substrate. Interaction of these groups with the ethereal hetero-atom can have a neutral, an additive or an antagonistic effect on transition state stabilization, depending on their molecular structure, size and enantiomeric configuration.     

Exploring the action and specificity of cobra venom phospholipase A2 toward human erythrocytes, ghost membranes, and lipid mixtures

We have investigated the action and substrate specificity of phospholipase A2 (EC 3.1.1.4) purified from cobra venom (Naja naja naja) toward intact and Triton-solubilized human erythrocytes, toward ghost membranes, and toward extracted ghost lipids in mixed micelles with Triton X-100. We have found that: (i) phospholipids in the outer surface of intact erythrocytes are extremely poor substrates for the phospholipase, (ii) phospholipids in ghost erythrocyte membranes and in Triton-solubilized erythrocytes are suitable substrates for the enzyme, (iii) in these latter systems which contain a mixture of lipids, phosphatidylethanolamine is preferentially hydrolyzed, whereas in model studies on individual phospholipid species in mixed micelles with Triton, phosphatidylcholine is the preferred substrate of the enzyme, and (iv) the preferential hydrolysis of phosphatidylethanolamine is also observed for extracted ghost lipid mixtures in mixed micelles. These results demonstrate a dependence of phospholipase A2 activity on the ghosting procedure and a dependence of substrate specificity on the presence of other lipids. The relevance of these findings to the interpretation of membrane lipid asymmetry studies utilizing phospholipases is considered in detail.     

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.