Computational analysis of the membrane association of group IIA secreted phospholipases A2: a differential role for electrostatics

Secreted phospholipases A2 (sPLA2's) are enzymes that hydrolyze glycerophospholipids at the sn-2 position, which leads to the production of lipid mediators of many cellular processes. These interfacial enzymes are regulated by their lipid specificity at two levels: membrane binding and substrate recognition. Different sPLA2's utilize different combinations of electrostatic and hydrophobic interactions to adsorb to membrane surfaces, which results in the wide range of membrane binding behaviors observed. Here, the finite difference Poisson Boltzmann (FDPB) method is used to quantitatively analyze the contribution of electrostatic interactions to the membrane association of two highly basic group II sPLA2's: Agkistrodon piscivorus piscivorus (AppD49) sPLA2 and nonpancreatic human group IIA (hGIIA) sPLA2. The calculations predict how membrane binding is affected by ionic strength, membrane composition, substitutions of residues in the enzymes, and the presence of calcium in the active site. In addition, the results provide molecular models for the membrane-associated forms of the enzymes. Furthermore, these models account for (1) changes in orientation and protonation state of both the native and charge reversal forms of the enzymes at the membrane surface and (2) the effect of protein/vesicle aggregation, as observed for hGIIA sPLA2. Importantly, the modeling quantitatively describes the complex membrane binding behaviors of these interfacial enzymes in terms of simple physical forces and provides structural information that is difficult to obtain experimentally. The computational analysis shows that nonspecific electrostatic interactions not only play a major role in recruiting these enzymes to membrane surfaces but also orient the enzymes for productive catalysis at the membrane interface.     

Molecular dynamics simulations of protein-tyrosine phosphatase 1B. II. substrate-enzyme interactions and dynamics

Molecular dynamics simulations of protein tyrosine phosphatase 1B (PTP1B) complexed with the phosphorylated peptide substrate DADEpYL and the free substrate have been conducted to investigate 1) the physical forces involved in substrate-protein interactions, 2) the importance of enzyme and substrate flexibility for binding, 3) the electrostatic properties of the enzyme, and 4) the contribution from solvation. The simulations were performed for 1 ns, using explicit water molecules. The last 700 ps of the trajectories was used for analysis determining enthalpic and entropic contributions to substrate binding. Based on essential dynamics analysis of the PTP1B/DADEpYL trajectory, it is shown that internal motions in the binding pocket occur in a subspace of only a few degrees of freedom. In particular, relatively large flexibilities are observed along several eigenvectors in the segments: Arg(24)-Ser(28), Pro(38)-Arg(47), and Glu(115)-Gly(117). These motions are correlated to the C- and N-terminal motions of the substrate. Relatively small fluctuations are observed in the region of the consensus active site motif (H/V)CX(5)R(S/T) and in the region of the WPD loop, which contains the general acid for catalysis. Analysis of the individual enzyme-substrate interaction energies revealed that mainly electrostatic forces contribute to binding. Indeed, calculation of the electrostatic field of the enzyme reveals that only the field surrounding the binding pocket is positive, while the remaining protein surface is characterized by a predominantly negative electrostatic field. This positive electrostatic field attracts negatively charged substrates and could explain the experimentally observed preference of PTP1B for negatively charged substrates like the DADEpYL peptide.     

Differential interfacial and substrate binding modes of mammalian pancreatic phospholipases A2: a comparison among human, bovine, and porcine enzymes.

To identify the residues essential for interfacial binding and substrate binding of human pancreatic phospholipase A2 (hpPLA2), several ionic residues in the putative interfacial binding surface (R6E, K7E, K10E, and K116E) and substrate binding site (D53K and K56E) were mutated. Interfacial affinity of these mutants was measured using anionic polymerized liposomes, and their enzymatic activity was measured using various substrates including phospholipid monomers, zwitterionic and anionic micelles, and anionic polymerized mixed liposomes. Similar mutations (R6E, K10E, K56E, and K116E) were made to porcine pancreatic phospholipase A2 (ppPLA2), and the properties of mutants were measured by the same methods. Results indicate that hpPLA2 and ppPLA2 have similar interfacial binding mechanisms in which cationic residues in the amino terminus and Lys-116 in the carboxy terminus are involved in binding to anionic lipid surfaces. Small but definite differences between the two enzymes were observed in overall interfacial affinity and activity and the effects of the mutations on interfacial enzyme activity. The interfacial binding of hpPLA2 and ppPLA2 is distinct from that of bovine pancreatic phospholipase A2 in that Lys-56 is involved in the interfacial binding of the latter enzyme. The unique phospholipid headgroup specificity of hpPLA2 derives from the presence of Asp-53 in the substrate binding site. This residue appears to participate in stabilizing electrostatic interactions with the cationic ethanolamine headgroup, hence the phosphatidylethanolamine preference of hpPLA2. Taken together, these studies reveal the similarities and the differences in the mechanisms by which mammalian pancreatic phospholipases A2 interact with lipid aggregates and perform interfacial catalysis.     

The interaction between the presynaptic phospholipase neurotoxins beta-bungarotoxin and crotoxin and mixed detergent-phosphatidylcholine micelles. A comparison with non-neurotoxic snake venom phospholipases A2

Certain phospholipase A2 enzymes (E.C.3.1.1.4) selectively inhibit neurotransmitter release from cholinergic nerve terminals. Both specific acceptor proteins and the physical state of nerve terminal phospholipids have been implicated in studies of the mechanism of phospholipase neurotoxin action. Here we have examined the effects of charge on a micellar phospholipid substrate by comparing the enzyme activity and binding of two neurotoxic phospholipases (beta-bungarotoxin and crotoxin) with other non-neurotoxic phospholipases. This has been achieved by altering either the phospholipid or the ionic charge of the detergent in the mixed phospholipid micelle. The neurotoxic phospholipases were only active on negatively charged micelles, whereas the non-neurotoxic enzymes were equally active in hydrolyzing neutral micelles. This distinction was also reflected in binding studies; the non-neurotoxic phospholipases bound to both types of substrate, whereas beta-bungarotoxin and crotoxin selectively bound to negatively charged micellar structures. These experiments suggest that, in addition to the existence of any specific acceptor proteins, neurotoxin binding is also governed by the charge on the lipid phase of the nerve terminal membrane.     

Membrane-perturbing activity of Viperidae myotoxins: an electrostatic surface potential approach to a puzzling problem

Phospholipase-like myotoxins are a class of proteins present in Viperidae venom. Despite the high level of amino acid and structural homology with soluble phospholipases A(2), myotoxins are devoid of enzymatic activity and share cytolytic activity by means of a totally unknown mechanism involving the lipid bilayer perturbation. The distribution of electrostatic surface potentials of four myotoxins and seven phospholipases A(2) has been compared. The charge distribution is similar in all active non-cytolytic phospholipases with a strongly positive side corresponding to the domain interacting with the micellar substrate and with the opposite side negatively charged. In contrast, all myotoxins examined are positively charged on both sides. Myotoxin III, the only known example of a myotoxin sharing enzymatic activity, displays the same electrostatic surface potential as other related toxins. Using liposomes made with non-hydrolysable phospholipids, we demonstrate that myotoxin III perturbs the lipid bilayer like other myotoxins. Based on these results, a molecular model for myotoxin-membrane perturbing activity is proposed. In this model, potential double-face binding of myotoxic phospholipases A(2) to lipid surfaces could trigger a lipid bilayer destabilization and could generate a stable fusion pore, probably because of the presence of hydrophobic moieties that flank the cationic sites.     

Action of phospholipases A2 on phosphatidylcholine bilayers. Effects of the phase transition, bilayer curvature and structural defects

We examined the action of porcine pancreatic and bee-venom phospholipase A2 towards bilayers of phosphatidylcholine as a function of several physical characteristics of the lipid-water interface. 1. Unsonicated liposomes of dimyristoyl phosphatidylcholine are degraded by both phospholipases in the temperature region of the phase transition only (cf. Op den Kamp et al. (1974) Biochim. Biophys. Acta 345, 253--256 and Op den Kamp et al. (1975) Biochim. Biophys. Acta 406, 169--177). With sonicates the temperature range in which hydrolysis occurs is much wider. This discrepancy between liposomes and sonicates cannot be ascribed entirely to differences in available substrate surface. 2. Below the phase-transition temperature the phospholipases degrade dimyristoyl phosphatidylcholine single-bilayer vesicles with a strongly curved surface much more effectively than larger single-bilayer vesicles with a relatively low degree of curvature. 3. Vesicles composed of egg phosphatidylcholine can be degraded by pancreatic phospholipase A2 at 37 degrees C, provided that the substrate bilayer is strongly curved. The bee-venom enzyme shows a similar, but less pronounced, preference for small substrate vesicles. 4. In a limited temperature region just above the transition temperature of the substrate the action of both phospholipases initially proceeds with a gradually increasing velocity. This stimulation is presumably due to an increase of the transition temperature, effectuated by the products of the phospholipase action. 5. Structural defects in the substrate bilayer, introduced by sonication below the phase-transition temperature (cf. Lawaczeck et al. (1976) Biochim. Biophys. Acta 443, 313--330) facilitate the action of both phospholipases. The results lead to the general conclusion that structural irregularities in the packing of the substrate molecules facilitate the action of phospholipases A2 on phosphatidylcholine bilayers. Within the phase transition and with bilayers containing structural defects these irregularities represent boundaries between separate lipid domains. The stimulatory effect of strong bilayer curvature can be ascribed to an overall perturbation of the lipid packing as well as to a change in the phase-transition temperature.     

Amino acid substitutions of the NH2-terminal Ala1 of porcine pancreatic phospholipase A2: a monolayer study

Previously it has been shown that the binding of porcine pancreatic phospholipase A2 to lipid-water interfaces is governed by the pK of the alpha-NH3+ group of the N-terminal alanine. Chemically modified phospholipases A2 in which the N-terminal Ala has been replaced by D-Ala or in which the polypeptide chain has been elongated with DL-Ala no longer display activity toward micellar substrate. The activity of DL-Ala-1-, [D-Ala1]-, and [Gly1]phospholipases A2 on substrate monolayers, which allow a continuous change in the packing density of the lipid molecule, was investigated. At pH 6 [Gly1]phospholipase A2 behaves like the native enzyme on lecithin monolayers. DL-Ala1- and [D-Ala1]phospholipases A2, although they are active in this system, showed a weaker lipid penetration capacity at this pH. Studies on the pH and Ca2+ ion dependency of the pre-steady-state kinetics and of the activity of these radiolabeled proteins showed that [D-Ala1]phospholipase A2 does not possess a second low-affinity site for Ca2+ ions in contrast to the native phospholipase A2. This second low-affinity Ca2+ binding site, which is also absent in [Gly1]phospholipase A2, is induced in the latter enzyme by the presence of lipid-water interfaces.     

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)     

Interfacial catalysis by phospholipase A2: monomeric enzyme is fully catalytically active at the bilayer interface.

Interfacial catalysis in the scooting mode by phospholipase A2 (PLA2) from pancreas and venoms (18 different preparations) was examined on vesicles of 1,2-dimyristoyl-sn-glycero-3-phosphomethanol under the conditions where the rates of transbilayer and intervesicle exchanges of the enzyme, substrate, and the products of hydrolysis were negligible on the time scale (less than 30 min) on which all the substrate molecules on the outer monolayer of the target vesicles were hydrolyzed. The reaction progress curves for all PLA2s exhibited no latency period (less than 3 s). When the vesicle to PLA2 ratio in the reaction mixture was high so that according to the Poissonian distribution model at most one enzyme was bound to a vesicle, the extent of hydrolysis increased linearly with the amount of enzyme in the reaction mixture. However, the extent of hydrolysis per enzyme, NS, remained the same for all PLA2s, and it corresponded to the size of the target vesicles determined by independent methods. Similarly, the initial rate of hydrolysis increased linearly with the enzyme concentration, and the slope of the log-log plot was one under the conditions of one or more enzyme per vesicle. Such observations showed that monomeric PLA2 is fully catalytically active at the interface. This conclusion was supported by the absence of intermolecular resonance energy transfer from tryptophan-3 donor in the native PLA2 to the anthraniloyl acceptor in An87-PLA2, the catalytically active derivative of PLA2 with an anthraniloyl fluorophore on lysine 87. In this system, intermolecular resonance energy transfer was seen only when the donor-acceptor molecules were "crowded" at a high surface density with a relatively low lipid to protein mole ratio. On the basis of these results, it was concluded that secretory PLA2s from venoms and pancreas are fully catalytically active as monomers. Additional studies reported here showed that acylation of PLA2 was not necessary for catalysis or binding to the interface and that the binding of the substrate to the active site of PLA2 was not necessary for the binding of the enzyme to the interface.     

Phospholipase A2 at the bilayer interface.

Interfacial catalysis is a necessary consequence for all enzymes that act on amphipathic substrates with a strong tendency to form aggregates in aqueous dispersions. In such cases the catalytic event occurs at the interface of the aggregated substrate, the overall turnover at the interface is processive, and it is influenced the molecular organization and dynamics of the interface. Such enzymes can access the substrate only at the interface because the concentration of solitary monomers of the substrate in the aqueous phase is very low. Moreover, the microinterface between the bound enzyme and the organized substrate not only facilitates formation of the enzyme-substrate complex, but a longer residence time of the enzyme at the substrate interface also promotes high catalytic processivity. Binding of the enzyme to the substrate interface as an additional step in the overall catalytic turnover permits adaptation of the Michaelis-Menten formalism as a basis to account for the kinetics of interfacial catalysis. As shown for the action of phospholipase A2 on bilayer vesicles, binding equilibrium has two extreme kinetic consequences. During catalysis in the scooting mode the enzyme does not leave the surface of the vesicle to which it is bound. On the other hand, in the hopping mode the absorption and desorption steps are a part of the catalytic turnover. In this minireview we elaborate on the factors that control binding of pig pancreatic phospholipase A2 to the bilayer interface. Binding of PLA2 to the interface occurs through ionic interactions and is further promoted by hydrophobic interactions which probably occur along a face of the enzyme, with a hydrophobic collar and a ring of cationic residues, through which the catalytic site is accessible to substrate molecules in the bilayer. An enzyme molecule binds to the surface occupied by about 35 lipid molecules with an apparent dissociation constant of less than 0.1 pM for the enzyme on anionic vesicles compared to 10 mM on zwitterionic vesicles. Results at hand also show that aggregation or acylation of the protein is not required for the high affinity binding or catalytic interaction at the interface.     

Electrostatic fields at the active site of ribulose-1,5-bisphosphate carboxylase.

A macroscopic approach has been employed to calculate the electrostatic potential field of nonactivated ribulose-1,5-bisphosphate carboxylase and of some complexes of the enzyme with activator and substrate. The overall electrostatic field of the L2-type enzyme from the photosynthetic bacterium Rhodospirillum rubrum shows that the core of the dimer, consisting of the two C-terminal domains, has a predominantly positive potential. These domains provide the binding sites for the negatively charged phosphate groups of the substrate. The two N-terminal domains have mainly negative potential. At the active site situated between the C-terminal domain of one subunit and the N-terminal domain of the second subunit, a large potential gradient at the substrate binding site is found. This might be important for polarization of chemical bonds of the substrate and the movement of protons during catalysis. The immediate surroundings of the activator lysine, K191, provide a positive potential area which might cause the pK value for this residue to be lowered. This observation suggests that the electrostatic field at the active site is responsible for the specific carbamylation of the epsilon-amino group of this lysine side chain during activation. Activation causes a shift in the electrostatic potential at the position of K166 to more positive values, which is reflected in the unusually low pK of K166 in the activated enzyme species. The overall shape of the electrostatic potential field in the L2 building block of the L8S8-type Rubisco from spinach is, despite only 30% amino acid homology for the L-chains, strikingly similar to that of the L2-type Rubisco from Rhodospirillum rubrum. A significant difference between the two species is that the potential is in general more positive in the higher plant Rubisco. In particular, the second phosphate binding site has a considerably more positive potential, which might be responsible for the higher affinity for the substrate of L8S8-type enzymes. The higher potential at this site might be due to two remote histidine residues, which are conserved in the plant enzymes.     

Monoclonal antibodies against rat platelet phospholipase A2

Monoclonal antibodies which bind specifically to rat platelet phospholipase A2 have been raised. None of them bound to exocrine phospholipase A2 derived from pancreas or snake venom. All antibodies recognized the conformational structure of rat platelet phospholipase A2 supported by intramolecular disulfide bonds, since the reactivity between the antibodies and the enzyme was lost in the presence of 2-mercaptoethanol. One of them, designed MB5.2, inhibited the activity of the platelet phospholipase A2 in a dose-dependent manner. A kinetic study revealed that antibody MB5.2 apparently competed with the substrate for the active site of the enzyme. The other antibodies, designed MD7.1 and ME6.1, inhibited the binding of the enzyme to heparin. The distribution of phospholipases A2 bearing a similar determinant to rat platelet phospholipase A2 was investigated by immunoprecipitation of the enzyme activity or by an immunoblot technique. Among rat tissues, cross-reactivity was observed with phospholipases A2 from spleen, lung, and bone marrow. Extracellular phospholipase A2 detected in the peritoneal cavity of casein-treated rat was also recognized by these antibodies. Furthermore, antibody MD7.1 cross-reacted with rabbit and guinea pig platelet phospholipases A2.     

Two different proteins that compete for binding to thrombin have opposite kinetic and thermodynamic profiles

Thrombin binds thrombomodulin (TM) at anion binding exosite 1, an allosteric site far from the thrombin active site. A monoclonal antibody (mAb) has been isolated that competes with TM for binding to thrombin. Complete binding kinetic and thermodynamic profiles for these two protein-protein interactions have been generated. Binding kinetics were measured by Biacore. Although both interactions have similar K(D)s, TM binding is rapid and reversible while binding of the mAb is slow and nearly irreversible. The enthalpic contribution to the DeltaG(bind) was measured by isothermal titration calorimetry and van't Hoff analysis. The contribution to the DeltaG(bind) from electrostatic steering was assessed from the dependence of the k(a) on ionic strength. Release of solvent H(2)O molecules from the interface was assessed by monitoring the decrease in amide solvent accessibility at the interface upon protein-protein binding. The mAb binding is enthalpy driven and has a slow k(d). TM binding appears to be entropy driven and has a fast k(a). The favorable entropy of the thrombin-TM interaction seems to be derived from electrostatic steering and a contribution from solvent release. The two interactions have remarkably different thermodynamic driving forces for competing reactions. The possibility that optimization of binding kinetics for a particular function may be reflected in different thermodynamic driving forces is discussed.     

Mechanism of gold nanoparticles-induced trypsin inhibition: a multi-technique approach

The binding interactions of gold nanoparticles with trypsin were investigated using multi-spectra methods and molecular modeling. The experiment data showed that trypsin modified the surface of gold nanoparticles. The fluorescence intensity of trypsin was quenched by gold nanoparticles that strongly associated with protein and induced the inhibition of enzyme activity. The electrostatic and hydrophobic interactions were the primary contributors to the binding forces between trypsin and gold nanoparticles. The covalent interactions might be also involved in the binding process. The modeling calculated results indicated that the binding site was near to the primary substrate-binding pocket and the active site of the enzyme substrate. This work elucidated the interaction mechanism of trypsin with gold nanoparticles from the theoretical and experimental angle.     

Effect of the lipid interface on the catalytic activity and spectroscopic properties of a fungal lipase

Lipase from the fungi Thermomyces (formerly Humicola) lanuginosa (TlL) is widely used in industry. This interfacial enzyme is inactive under aqueous conditions, but catalytic activation is induced on binding to a lipid-water interface. In order for protein engineering to design more efficient mutants of TlL for specific applications, it is important to characterize its interfacial catalysis. A complete analysis of steady-state kinetics for the hydrolysis of a soluble substrate by TlL has been developed using an interface different from the substrate. Small vesicles of 1-palmitoyl-2-oleoylglycero-sn-3-phosphoglycerol (POPG) or other anionic phospholipids are a neutral diluent interface for the partitioning of substrate and enzyme. TlL binds to these interfaces in an active or open form, thus implying a displacement of the helical lid away from the active site. A study of the influence of substrate and diluent concentration dependence of the rate of hydrolysis provides a basis for the determination of the primary interfacial catalytic parameters. The interfacial activation is not supported by zwitterionic vesicles or by large anionic vesicles of 100 nm diameter, although TlL binds to these interfaces. Using a combination of fluorescence-based techniques applied to several mutants of TlL with different tryptophan residues we have shown that TlL binds to phospholipid vesicles in different forms rendering different catalytic activities, and that the open lid conformation is achieved and stabilized by a combination of electrostatic and hydrophobic interactions between the enzyme's lipid-binding face and the interface.     

Substrate modulation of enzyme activity in the herpesvirus protease family

The herpesvirus proteases are an example in which allosteric regulation of an enzyme activity is achieved through the formation of quaternary structure. Here, we report a 1.7 A resolution structure of Kaposi's sarcoma-associated herpesvirus protease in complex with a hexapeptide transition state analogue that stabilizes the dimeric state of the enzyme. Extended substrate binding sites are induced upon peptide binding. In particular, 104 A2 of surface are buried in the newly formed S4 pocket when tyrosine binds at this site. The peptide inhibitor also induces a rearrangement of residues that stabilizes the oxyanion hole and the dimer interface. Concomitant with the structural changes, an increase in catalytic efficiency of the enzyme results upon extended substrate binding. A nearly 20-fold increase in kcat/KM results upon extending the peptide substrate from a tetrapeptide to a hexapeptide exclusively due to a KM effect. This suggests that the mechanism by which herpesvirus proteases achieve their high specificity is by using extended substrates to modulate both the structure and activity of the enzyme.     

The lysine-49 phospholipase A2 from the venom of Agkistrodon piscivorus piscivorus. Relation of structure and function to other phospholipases A2

A new class of phospholipases A2 that have a lysine at position 49 differ from the more conventional Asp-49 enzymes with respect to the sequential binding of the essential cofactor, calcium, and the substrate, phospholipid, in the formation of the catalytic complex (Maraganore, J.M., Merutka, G., Cho, W., Welches, W., Kézdy, F.J., and Heinrikson, R.L. (1984) J. Biol. Chem. 259, 13839-13843). We report here the complete amino acid sequence of the Lys-49 enzyme from Agkistrodon piscivorus piscivorus. The sequence was determined by automated Edman degradation of the intact, S-carboxymethylcysteinyl protein and of peptides derived therefrom by cleavage with cyanogen bromide, chymotrypsin, trypsin, and endoproteinase Lys-C. Despite several changes at amino acid residues previously considered to be invariant, the Lys-49 enzymes are homologous to the Asp-49 phospholipases. Homology is especially apparent in the following: 1) the pattern of 14 half-cystine residues, 2) conservation of hydrophobic residues which have been shown to encircle the active site, and 3) conservation of Asp-99 and His-48 which have been implicated in the catalytic reaction itself. These observations together with kinetic and binding data imply that the Lys-49 phospholipases have a catalytic mechanism and a three-dimensional architecture similar to those of the Asp-49 enzymes. Modeling of the Lys-49 enzyme based upon the structure of bovine pancreatic phospholipase reveals that the epsilon-amino group of Lys-49 can fit easily in the calcium-binding site and, moreover, that this orientation of a cationic side chain at position 49 could account for the characteristic and novel feature of the Lys-49 phospholipases, i.e. that they are able to form complexes with phospholipid in the absence of calcium.     

Kinetic analysis of phospholipase A2 activity toward mixed micelles and its implications for the study of lipolytic enzymes.

A detailed kinetic scheme is proposed for the action of phospholipase A2 on mixed micelles of phospholipid and surfactant: see article. where E is the enzyme, A is the mixed micelle, and B is the phospholipid substrate in the mixed micelle. This scheme takes into account quantitatively the involvement of the lipid-water interface in the action of this enzyme toward substrate in macromolecular lipid complexes. The kinetic equation for this scheme is derived and four simplifying assumptions which are necessary for its practical application are described. Kinetic data are reported for the action of cobra venom phospholipase A2 (Naja naja naja) on 1,2-dipalmitosyl-sn-glycero-3-phosphorylcholine in mixed micelles with the nonionic surfactant Triton X-100, and these data are analyzed in terms of the kinetic equation presented. At 40 degrees, pH 8.0, and in the presence of 10 mM Ca2+, V was found to be about 4 X 10(3) mumol min(-1) mg of protein(-1). KsA, which is the dissociation constant for the enzyme-mixed micelle complex, is about 5 X 10(-4) M. KmB, the Michaelis constant for the catalytic step, which is (k-2 + k3)/k2, is 1 to 2 X 10(-10) mol cm-2. This kinetic treatment, together with the fact that the mixed micelle system allows the concentration of the substrate in the lipid-water interface to be varied, has made possible the quantitative separation of the association of a lipolytic enzyme with the lipid-water interface (expressed as KsA) and the binding to the substrate in the interface (reflected in the KmB term). The implications of this kinetic scheme for the analysis of phospholipase A2 from other sources acting on other aggregated forms of phospholipid and for the study of other phospholipases and lipases is considered.     

Choline acetyltransferase structure reveals distribution of mutations that cause motor disorders

Choline acetyltransferase (ChAT) synthesizes acetylcholine in neurons and other cell types. Decreases in ChAT activity are associated with a number of disease states, and mutations in ChAT cause congenital neuromuscular disorders. The crystal structure of ChAT reported here shows the enzyme divided into two domains with the active site in a solvent accessible tunnel at the domain interface. A low-resolution view of the complex with one substrate, coenzyme A, defines its binding site and suggests an additional interaction not found in the related carnitine acetyltransferase. Also, the preference for choline over carnitine as an acetyl acceptor is seen to result from both electrostatic and steric blocks to carnitine binding at the active site. While half of the mutations that cause motor disorders are positioned to affect enzyme activity directly, the remaining changes are surprisingly distant from the active site and must exert indirect effects. The structure indicates how ChAT is regulated by phosphorylation and reveals an unusual pattern of basic surface patches that may mediate membrane association or macromolecular interactions.     

The chemical basis for interfacial activation of monomeric phospholipases A2. Autocatalytic derivatization of the enzyme by acyl transfer from substrate

A basic monomeric phospholipase A2 from the venom of the American water moccasin, Agkistrodon piscivorus piscivorus, undergoes Ca2+-dependent, autocatalytic acylation during the course of hydrolysis of both model and natural phospholipid substrates. Acylation occurs at 2 lysine residues, Lys-7 and Lys-10, in the NH2-terminal alpha-helical segment of the enzyme, and when both positions are fully derivatized, the stable bisacylphospholipase A2 becomes a dimer in solution. The acylated enzyme is fully activated toward monomolecular layers of lecithins. Similar studies applied to the monomeric phospholipases A2 from porcine pancreas and from the venom of Agkistrodon contortrix contortrix also showed irreversible activation of the enzymes by substrate with the same kinetic consequences and formation of dimers. Acylation thus enables these enzymes to overcome the lag period observed under such conditions with native monomeric phospholipases, a phenomenon referred to as interfacial activation. Activation of the enzyme by acylation potentiates the phospholipase for interfacial recognition via formation of a dimeric enzyme. The naturally occurring phospholipase A2 dimer from Crotalus atrox venom displays no lag in the hydrolysis of lecithin monolayers nor does it undergo substrate level acylation. These facts support our proposal that dimerization concomitant with acylation is responsible for the large rate enhancements seen in the hydrolysis of aggregated phospholipids by monomeric phospholipases. Our findings demonstrate for the first time a chemical mechanism for interfacial activation of and interfacial recognition by phospholipases A2.