Interfacial control of lid opening in Thermomyces lanuginosa lipase

Small unilamelar vesicles of anionic phospholipids (SUV), such as 1-palmitoyl-2-oleoylglycero-sn-3-phosphoglycerol (POPG), provide an interface where Thermomyces lanuginosa triglyceride lipase (TlL) binds and adopts a catalytically active conformation for the hydrolysis of substrate partitioned in the interface, such as tributyrin or p-nitrophenylbutyrate, with an increase in catalytic rate of more than 100-fold for the same concentration of substrate [Berg et al. (1998) Biochemistry 37, 6615-6627.]. This interfacial activation is not seen with large unilamelar vesicles (LUV) of the same composition, or with vesicles of zwitterionic phospholipids such as 1-palmitoyl-2-oleoylglycero-sn-3-phosphocholine (POPC), independently of the vesicle size. Tryptophan fluorescence experiments show that lipase binds to all those types of vesicles with similar affinity, but it adopts different forms that can be correlated with the enzyme catalytic activity. The spectral change on binding to anionic SUV corresponds to the catalytically active, or "open" form of the enzyme, and it is not modified in the presence of substrate partitioned in the vesicles, as demonstrated with inactive mutants. This indicates that the displacement of the lid characteristic of lipase interfacial activation is induced by the anionic phospholipid interface without blocking the accessibility of the active site to the substrate. Experiments with a mutant containing only Trp89 in the lid show that most of the spectral changes on binding to POPG-SUVs take place in the lid region that covers the active site; an increase in Trp anisotropy indicates that the lid becomes less flexible in the active form, and quenching experiments show that it is significantly buried from the aqueous phase. On the other hand, results with a mutant where Trp89 is changed to Leu show that the environment of the structural tryptophans in positions 117, 221, and 260 is somehow altered on binding, although their mobility and solvent accessibility remains the same as in the inactive form in solution. The form of TlL bound to POPC-SUV or -LUV vesicles as well as to LUV vesicles of POPG has the same spectral signatures and corresponds to an inactive or "closed" form of the enzyme. In these interfaces, the lid is highly flexible, and Trp89 remains accessible to solvent. Resonance energy transfer experiments show that the orientation of TlL in the interface is different in the active and inactive forms. A model of interaction consistent with these data and the available X-ray structures is proposed. This is a unique system where the composition and physical properties of the lipid interface control the enzyme activity.     

Effects of a fungal lipase on membrane organization evaluated by fluorescence polarization

Triglyceride lipase from Thermomyces lanuginosa (TlL) binds to the non-substrate small (40 nm) unilamelar vesicles of 1,2-dimiristoylglycero-sn-3-phosphoglycerol (DMPG-SUVs) in a catalytically active structure, whereas it adopts a catalytically incompetent form in binding to zwitterionic 1,2-dimiristoylglycero-sn-3-phosphocholine (DMPC-SUVs) or to large (100 nm) unilamelar DMPG (DMPG-LUVs) vesicles. Steady-state anisotropy measurements with probes that localize at different positions in the membrane give information on the effects of TlL (and its mutants) on the mobility of the phospholipids. All TlL mutants insert into the DMPG-SUVs and increase lipid order at the headgroup region and at the hydrophobic core of the lipid bilayer as well. The increase of the rigidity of the membrane that occurs in the gel and liquid crystal states, results in an increase of the phase transition temperature (T_m). Kinetic experiments with monolayers of 1,2-dicaprin demonstrate the thermal stability of the enzyme in the range of temperatures of the phase transition. Mutations in the tryptophan (Trp) residues of TlL reduce activity of this enzyme and affect its interaction with the membrane. The membrane insertions of TlL mutants with other than Trp substitutions are much more shallower and produce only small increases of T_m, whereas mutation of lid-located Trp89 or mutation of any other Trp residue (117, 221, 260) result in a deeper penetration and significant increases of the T_m. Lipid dynamics of DMPC-SUVs or DMPG-LUVs are not affected by any of the TlL mutants, despite their strong binding to the lipids revealed by resonance energy transfer (RET). These results are consistent with the lipase–lipid penetration model in which the “lid” region of TlL inserts into the highly curved anionic interface, thus stabilizing the “open” or active enzyme conformation, whereas TlL binds to the surface of zwitterionic and large (small curvature) anionic vesicles in a “closed” (or inactive) conformation, without insertion of the lid.     

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.     

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.     

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.     

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.     

Optimizing the interfacial binding and activity of a bacterial phosphatidylinositol-specific phospholipase C

The phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis can be activated by nonsubstrate interfaces such as phosphatidylcholine micelles or bilayers. This activation corresponds with partial insertion into the interface of two tryptophans, Trp-47 in helix B and Trp-242 in a loop, in the rim of the alphabeta-barrel. Both W47A and W242A have much weaker binding to interfaces and considerably lower kinetic interfacial activation. Tryptophan rescue mutagenesis, reinsertion of a tryptophan at a different place in helix B in the W47A mutant or in the loop (residues 232-244) of the W242A mutant, has been used to determine the importance and orientation of a tryptophan in these two structural features. Phosphotransferase and phosphodiesterase assays, and binding to phosphatidylcholine vesicles were used to assess both orientation and position of tryptophans needed for interfacial activity. Of the helix B double mutants, only one mutant, I43W/W47A, has tryptophan in the same orientation as Trp-47. I43W/W47A shows recovery of phosphatidylinositol-specific phospholipase C (PC) activation of d-myo-inositol 1,2-cyclic phosphate hydrolysis. However, the specific activity toward phosphatidylinositol is still lower than wild type enzyme and high activity with phosphatidylinositol solubilized in 30% isopropyl alcohol (a hallmark of the native enzyme) is lost. Reinserting a tryptophan at several positions in the loop composed of residues 232-244 partially recovers PC activation and affinity of the enzyme for lipid interfaces as well as activation by isopropyl alcohol. G238W/W242A shows an enhanced activation and affinity for PC interfaces above that of wild type. These results provide constraints on how this bacterial phosphatidylinositol-specific phospholipase C binds to activating PC interfaces.     

Control of the chemical step by leucine-31 of pancreatic phospholipase A2

A well-defined region of pancreatic and other secreted phospholipase A2 (PLA2), which we call the i-face, makes a molecular contact with the interface to facilitate and control the events and processivity of the interfacial catalytic turnover cycles. The structural features of the i-face and its allosteric relationship to the active site remain to be identified. As a part of the calcium binding (26-34) loop, Leu-31 is located on the surface near the substrate binding slot of PLA2. Analysis of the primary rate and equilibrium parameters of the Leu-31 substitution mutants of the pig pancreatic PLA2 shows that the only significant effect of the substitution is to impair the chemical step at the zwitterionic interface in the presence of added NaCl, and only a modest effect is seen on kcat at the anionic interface. Leu-31 substitutions have little effect on the binding of the enzyme to the interface; the affinity for certain substrate mimics is modestly influenced in W3F, L31W double mutant. The fluorescence emission results with the double mutant show that the microenvironment of Trp-31 is qualitatively different at the zwitterionic versus anionic interfaces. At both of the interfaces Trp-31 is not shielded from the bulk aqueous environment as it remains readily accessible to acrylamide and water. The NaCl-induced change in the Trp-31 emission spectrum of the double mutant on the zwitterionic interface is similar to that seen on the binding to the anionic interface. Together, the kinetic and spectroscopic results show that the form of PLA2 at the zwitterionic interface (Ez) is distinguishably different from the catalytically more efficient form at the anionic interface (Ea). This finding provides a structural basis for the two-state model for kcat activation by the anionic interface. In conjunction with earlier results we suggest that neutralization of certain cationic residues of PLA2 exerts a control on the calcium loop through residue 31.     

Activation, inhibition, and destabilization of Thermomyces lanuginosus lipase by detergents

Lipases catalyze the hydrolysis of triglycerides and are activated at the water-lipid interface. Thus, their interaction with amphiphiles such as detergents is relevant for an understanding of their enzymatic mechanism. In this study, we have characterized the effect of nonionic, anionic, cationic, and zwitterionic detergents on the enzymatic activity and thermal stability of Thermomyces lanuginosus lipase (TlL). For all detergents, low concentrations enhance the activity of TlL toward p-nitrophenyl butyrate by more than an order of magnitude; at higher detergent concentrations, the activity declines, leveling off close to the value measured in the absence of detergent. Surprisingly, these phenomena mainly involve monomeric detergent, as activation and inhibition occur well below the cmc for the nonionic and zwitterionic detergents. For anionic and cationic detergents, activation straddles the monomer-micelle transition. The data can be fitted to a three state interaction model, comprising free TlL in the absence of detergent, an activated complex with TlL at low detergent concentrations, and an enzyme-inhibiting complex at higher concentrations. For detergents with the same headgroup, there is an excellent correspondence between carbon chain length and ability to activate and inhibit TlL. However, the headgroup and number of chains also modulate these effects, dividing the detergents overall into three broad groups with rising activation and inhibition ability, namely, anionic and cationic detergents, nonionic and single-chain zwitterionic detergents, and double-chain zwitterionic detergents. As expected, only anionic and cationic detergents lead to a significant decrease in lipase thermal stability. Since nonionic detergents activate TlL without destabilizing the protein, activation/inhibition and destabilization must be independent processes. We conclude that lipase-detergent interactions occur at many independent levels and are governed by a combination of general and structurally specific interactions. Furthermore, activation of TlL by detergents apparently does not involve the classical interfacial activation phenomenon as monomeric detergent molecules are in most cases responsible for the observed increase in activity.     

Effect of the structure of phospholipid on the kinetics of intravesicle scooting of phospholipase A2

Action of pig pancreatic phospholipase A2 on vesicles of over 50 synthetic 1,2-diacylglycerol-3-phosphate derivatives and analogs is examined in the absence of any additives. In general, shorter acyl chains and small substituents on the phosphate make a better substrate, while phospholipids with large apolar substituents are not hydrolyzed. The interfacial turnover rate constant for scooting kinetics, ki, for the various phospholipids were from less than 0.1 to 1 per min. Intervesicle exchange of the bound enzyme is faster in vesicles of phospholipids with larger polar substituents, and it is promoted in the presence of anions like chloride, sulfate and thiocyanate. These factors lower the residence time of the enzyme on the bilayer and therefore effectively decrease the rate of hydrolysis. The apparent Km for the enzyme in the interface of anionic phospholipids in the presence of salts is in the 40 to 100 microM range which is 3- to 7-times larger than the dissociation constants for the bound enzyme measured by fluorescence enhancement of Trp-3. The quantum yield of the bound enzyme in vesicles of the various lipids is found to be up to 4-fold different. It is suggested that this difference is due to the E* + S to E*S equilibrium, where E*S has higher fluorescence intensity. The role of calcium in generating the enzyme binding site at the anionic interface, the role of anion anchoring site on the enzyme, and the relationship between the catalytic efficiency and the fluorescence quantum yields are discussed.     

On the binding preference of human groups IIA and X phospholipases A2 for membranes with anionic phospholipids

Mammals contain 9-10 secreted phospholipases A(2) (sPLA(2)s) that display widely different affinities for membranes, depending on the phospholipid composition. The much higher enzymatic activity of human group X sPLA(2) (hGX) compared with human group IIA sPLA(2) (hGIIA) on phosphatidylcholine (PC)-rich vesicles is due in large part to the higher affinity of the former enzyme for such vesicles; this result also holds when vesicles contain cholesterol and sphingomyelin. The inclusion of anionic phosphatidylserine in PC vesicles dramatically enhances interfacial binding and catalysis of hGIIA but not of hGX. This is the result of the large number of lysine and arginine residues scattered over the entire surface of hGIIA, which cause the enzyme to form a supramolecular aggregate with multiple vesicles. Thus, high affinity binding of hGIIA to anionic vesicles is a complex process and cannot be attributed to a few basic residues on its interfacial binding surface, as is also evident from mutagenesis studies. The main reason hGIIA binds poorly to PC-rich vesicles is that it lacks a tryptophan residue on its interfacial binding surface, a residue that contributes to the high affinity binding of hGX to PC-rich vesicles. Results show that the lag in the onset of hydrolysis of PC vesicles by hGIIA is due in part to the poor affinity of this enzyme for these vesicles. Binding affinity of hGIIA, hGX, and their mutants to PC-rich vesicles is well correlated to the ability of these enzymes to act on the PC-rich outer plasma membrane of mammalian cells.     

Effect of tryptophan insertions on the properties of the human group IIA phospholipase A2: mutagenesis produces an enzyme with characteristics similar to those of the human group V phospholipase A2

An important characteristic of the human group IIA secreted phospholipase A(2) (IIA PLA(2)) is the extremely low activity of this enzyme with phosphatidylcholine (PC) vesicles, mammalian cell membranes, and serum lipoproteins. This characteristic is reflected in the lack of ability of this enzyme to bind productively to zwitterionic interfaces. Part of the molecular basis for this lack of activity is an absence of tryptophan, a residue with a known preference for residing in the interfacial region of zwitterionic phospholipid bilayers. In this paper we have replaced the eight residues that make up the hydrophobic collar on the interfacial binding surface of the enzyme with tryptophan. The catalytic and interfacial binding properties of these mutants have been investigated, particularly those properties associated with binding to and hydrolysis of zwitterionic interfaces. Only the insertion of a tryptophan at position 3 or 31 produces mutants that significantly enhance the activity of the human IIA enzyme against zwitterionic interfaces and intact cell membranes. Importantly, the ability of the enzyme mutants to hydrolyze PC-rich interfaces such as the outer plasma membrane of mammalian cells was paralleled by enhanced interfacial binding to zwitterionic interfaces. The corresponding double tryptophan mutant (V3,31W) displays a specific activity on PC vesicles comparable to that of the human group V sPLA2. This enhanced activity includes the ability to interact with human embryonic kidney HEK293 cells, previously reported for the group V enzyme [Kim, Y. J., Kim, K. P., Rhee, H. J., Das, S., Rafter, J. D., Oh, Y. S., and Cho, W. (2002) J. Biol. Chem. 277, 9358-9365].     

Implications of surface charge and curvature for the binding orientation of Thermomyces lanuginosus lipase on negatively charged or zwitterionic phospholipid vesicles as studied by ESR spectroscopy

The triglyceride lipase (EC 3.1.1.3) Thermomyces lanuginosus lipase (TLL) binds with high affinity to unilamellar phospholipid vesicles that serve as a diluent interface for both lipase and substrate, but it displays interfacial activation on only small and negatively charged such vesicles [Cajal, Y., et al. (2000) Biochemistry 39, 413-423]. The productive-mode binding orientation of TLL at the lipid-water interface of small unilamellar vesicles (SUV) consisting of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG) was previously determined using electron spin resonance (ESR) spectroscopy in combination with site-directed spin-labeling [Hedin, E. M. K., et al. (2002) Biochemistry 41, 14185-14196]. In our investigation, we have studied the interfacial orientation of TLL when bound to large unilamellar vesicles (LUV) consisting of POPG, and bound to SUV consisting of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC). Eleven single-cysteine TLL mutants were spin-labeled as previously described, and studied upon membrane binding using the water soluble spin-relaxation agent chromium(III) oxalate (Crox). Furthermore, dansyl-labeled vesicles revealed the intermolecular fluorescence quenching efficiency between each spin-label positioned on TLL, and the lipid membrane. ESR exposure and fluorescence quenching data show that TLL associates closer to the negatively charged PG surface than the zwitterionic PC surface, and binds to both POPG LUV and POPC SUV predominantly through the concave backside of TLL opposite the active site, as revealed by the contact residues K74C-SL, R209C-SL, and T192C-SL. This orientation is significantly different compared to that on the POPG SUV, and might explain the differences in activation of the lipase. Evidently, both the charge and accessibility (curvature) of the vesicle surface determine the TLL orientation at the phospholipid interface.     

Roles of catalytic domain residues in interfacial binding and activation of group IV cytosolic phospholipase A2

Group IV cytosolic phospholipase A(2) (cPLA(2)) has been shown to play a critical role in eicosanoid biosynthesis. cPLA(2) is composed of the C2 domain that mediates the Ca(2+)-dependent interfacial binding of protein and the catalytic domain. To elucidate the mechanism of interfacial activation of cPLA(2), we measured the effects of mutations of selected ionic and hydrophobic residues in the catalytic domain on the enzyme activity and the membrane binding of cPLA(2). Mutations of anionic residues located on (Glu(419) and Glu(420)) or near (Asp(436), Asp(438), Asp(439), and Asp(440)) the active site lid enhanced the affinity for cPLA(2) for anionic membranes, implying that the electrostatic repulsion between these residues and the anionic membrane surface might trigger the opening of the active site. This notion is further supported by a biphasic dependence of cPLA(2) activity on the anionic lipid composition of the vesicles. Mutations of a cluster of cationic residues (Lys(541), Lys(543), Lys(544), and Arg(488)), while significantly enhancing the activity of enzyme, abrogated the specific activation effect by phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)). These data, in conjunction with cell activity of cPLA(2) and mutants transfected into HEK293 cells, suggest that the cationic residues form a specific binding site for PtdIns(4,5)P(2) and that the specific PtdIns(4,5)P(2) binding is involved in cellular activation of cPLA(2). Also, three hydrophobic residues at the rim of the active site (Ile(399), Leu(400), and Leu(552)) were shown to partially penetrate the membrane, thereby promoting membrane binding and activation of cPLA(2). Based on these results, we propose an interfacial activation mechanism for cPLA(2) which involves the removal of the active site lid by nonspecific electrostatic repulsion, the interdomain hinge movement induced by specific PtdIns(4,5)P(2) binding, and the partial membrane penetration by catalytic domain hydrophobic residues.     

Open and closed states of Candida antarctica lipase B: protonation and the mechanism of interfacial activation

Lipases (EC 3.1.1.3) are ubiquitous hydrolases for the carboxyl ester bond of water-insoluble substrates, such as triacylglycerols, phospholipids, and other insoluble substrates, acting in aqueous as well as in low-water media, thus being of considerable physiological significance with high interest also for their industrial applications. The hydrolysis reaction follows a two-step mechanism, or “interfacial activation,” with adsorption of the enzyme to a heterogeneous interface and subsequent enhancement of the lipolytic activity. Among lipases, Candida antarctica lipase B (CALB) has never shown any significant interfacial activation, and a closed conformation of CALB has never been reported, leading to the conclusion that its behavior was due to the absence of a lid regulating the access to the active site. The lid open and closed conformations and their protonation states are observed in the crystal structure of CALB at 0.91 Å resolution. Having the open and closed states at atomic resolution allows relating protonation to the conformation, indicating the role of Asp145 and Lys290 in the conformation alteration. The findings explain the lack of interfacial activation of CALB and offer new elements to elucidate this mechanism, with the consequent implications for the catalytic properties and classification of lipases.     

Interfacial catalysis by phospholipase A2: dissociation constants for calcium, substrate, products, and competitive inhibitors.

Interpretation of the kinetics of interfacial catalysis in the scooting mode as developed in the first paper of this series [Berg et al. (1991) Biochemistry 30 (first paper of six in this issue)], was based on the binding equilibrium for a ligand to the catalytic site of phospholipase A2. In this paper, we describe direct methods to determine the value of the Michaelis-Menten constant (KMS) for the substrate, as well as the equilibrium dissociation constants for ligands (KL) such as inhibitors (KI), products (KP), calcium (KCa), and substrate analogues (KS) bound to the catalytic site of phospholipase A2 at the interface. The KL values were obtained by monitoring the susceptibility to alkylation of His-48 at the catalytic site of pig pancreatic PLA2 bound to micellar dispersions of the neutral diluent 2-hexadecyl-sn-glycero-3-phosphocholine. The binding of the enzyme to dispersions of this amphiphile alone had little effect on the inactivation rate. The half-time for inactivation of the enzyme bound to micelles of the neutral diluent depended not only on the nature of the alkylating agent but also on the structure and the mole fraction of other ligands at the interface. The KL values for ligands obtained from the protection studies were in excellent accord with those obtained by monitoring the activation or inhibition of hydrolysis of vesicles of 1,2-dimyristoyl-sn-glycerophosphomethanol. Since only calcium, competitive inhibitors, and substrate analogues protected phospholipase A2 from alkylation, this protocol offered an unequivocal method to discern active-site-directed inhibitors from nonspecific inhibitors of PLA2, such as local anesthetics, phenothiazines, mepacrine, peptides related to lipocortin, 7,7-dimethyleicosadienoic acid, quinacrine, and aristolochic acid, all of which did not have any effect on the kinetics of alkylation nor did they inhibit the catalysis in the scooting mode.     

On the issue of interfacial activation of lipase in nonaqueous media

The question of whether lipases can be activated by adsorption onto an interface in organic solvents was addressed using Rhizomucor miehei lipase as a model. In aqueous solution, this enzyme was shown to undergo a marked interfacial activation. However, lipase (either lyophilized or precipitated from water with acetone) suspended in ethanol or 2-(2-ethoxyethoxy)ethanol containing triolein exhibited no jump in catalytic activity when the concentration of triolein exceeded its solubility in these solvents, thereby resulting in formation of an interface. To test whether the lack of interfacial activation was due to the insolubility of the enzyme in organic media, lipase was covalently modified with poly(ethylene glycol). The modified lipase, although soluble in nonaqueous media, was still unable to undergo interfacial activation, regardless of the hydrophobicity of the interface. This inability was found to be caused by the absence of adsorption of lipase onto interfaces in organic solvents, presumably because of the absence of the hydrophobic effect (the driving force of lipase adsorption onto hydrophobic interfaces in water) in such media. The uncovered lack of interfacial adsorption and activation suggests that the short alpha-helical "lid" covering the active center of the lipase remains predominantly closed in nonaqueous media, thus contributing to diminished enzymatic activity. (c) 1996 John Wiley & Sons, Inc.     

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

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

A Model for the Interfacial Kinetics of Phospholipase D Activity on Long-Chain Lipids

The membrane-active enzyme phospholipase D (PLD) catalyzes the hydrolysis of the phosphodiester bond in phospholipids and plays a critical role in cell signaling. This catalytic reaction proceeds on lipid-water interfaces and is an example of heterogeneous catalysis in biology. Recently we showed that planar lipid bilayers, a previously unexplored model membrane for these kinetic studies, can be used for monitoring interfacial catalytic reactions under well-defined experimental conditions with chemical and electrical access to both sides of the lipid membrane. Employing an assay that relies on the conductance of the pore-forming peptide gramicidin A to monitor PLD activity, the work presented here reveals the kinetics of hydrolysis of long-chain phosphatidylcholine lipids in situ. We have developed an extension of a basic kinetic model for interfacial catalysis that includes product activation and substrate depletion. This model describes the kinetic behavior very well and reveals two kinetic parameters, the specificity constant and the interfacial quality constant. This approach results in a simple and general model to account for product accumulation in interfacial enzyme kinetics.     

Importance of the lid and cap domains for the catalytic activity of gastric lipases

Human gastric lipase (HGL) is an enzyme secreted by the stomach, which is stable and active despite the highly acidic environment. It has been clearly established that this enzyme is responsible for 30% of the fat digestion processes occurring in human. This globular protein belongs to the alpha/beta hydrolase fold family and its catalytic serine is deeply buried under a domain called the extrusion domain, which is composed of a 'cap' domain and a segment consisting of 58 residues, which can be defined as a lid. The exact roles played by the cap and the lid domains during the catalytic step have not yet been elucidated. We have recently solved the crystal structure of the open form of the dog gastric lipase in complex with a covalent inhibitor. The detergent molecule and the inhibitor were mimicking a triglyceride substrate that would interact with residues belonging to both the cap and the lid domains. In this study, we have investigated the role of the cap and the lid domains, using site-directed mutagenesis procedures. We have produced truncated mutants lacking the lid and the cap. After expressing these mutants and purifying them, their activity was found to have decreased drastically in comparison with the wild type HGL. The lid and the cap domains play an important role in the catalytic reaction mechanism. Based on these results and the structural data (open form of DGL), we have pointed out the cap and the lid residues involved in the binding with the lipidic substrate.