Inhibition of pancreatic lipase in vitro by the covalent inhibitor tetrahydrolipstatin.

Tetrahydrolipstatin inhibits pancreatic lipase from several species, including man, with comparable potency. The lipase is progressively inactivated through the formation of a long-lived covalent intermediate, probably with a 1:1 stoichiometry. The lipase substrate triolein and also a boronic acid derivative, which is presumed to be a transition-state-form inhibitor, retard the rate of inactivation. Therefore, in all probability, tetrahydrolipstatin reacts with pancreatic lipase at, or near, the substrate binding or active site. Tetrahydrolipstatin is a selective inhibitor of lipase; other hydrolases tested were at least a thousand times less potently inhibited.     

Importance of the different steps of glycosylation for the activity and secretion of lipoprotein lipase in rat preadipocytes studied with monensin and tunicamycin

Lipoprotein lipase synthesized by cultured rat preadipocytes is present in three compartments: an intracellular, a surface-related 3-min heparin-releasable, and that secreted into the culture medium. 30 min after addition of 6 microM monensin, the lipoprotein lipase activity in the heparin-releasable compartment starts to decrease; by 4 h of monensin treatment the lipoprotein lipase activity in the heparin-releasable pool and in the culture medium is about 10% of that found in control dishes. The intracellular activity, which had been identified as lipoprotein lipase by an antiserum to lipoprotein lipase, increases slowly and doubles by 24 h. However, since the cellular compartment accounts for 10-25% of total activity, this increase does not account for the missing enzyme activity. To determine whether this enzyme molecule is synthesized but is not active, incorporation of labeled leucine, mannose and galactose into immunoadsorbable lipoprotein lipase was studied in control, monensin- or tunicamycin-treated cells. Addition of tunicamycin (5 micrograms/ml) for 24 h caused a 30-50% reduction in immunoadsorbable lipoprotein lipase, but the enzyme activity was reduced by 90%. On the other hand, 4 h monensin treatment reduced both incorporation of [3H]leucine into immunoadsorbable lipoprotein lipase and heparin-releasable and medium lipoprotein lipase activity by 57 to 77%. The immunoadsorbable lipoprotein lipase in the intracellular compartment has a [14C]mannose to [3H]galactose ratio of 0.15 and this ratio increased 6-fold in monensin-treated cells. The intracellular lipoprotein lipase in monensin-treated cells had the same affinity for both the native and synthetic substrate as the lipoprotein lipase in control cells, yet its spontaneous secretion into the culture medium and its release by 3 min heparin treatment was markedly decreased. The present results indicate that: the presence of asparagine-linked oligosaccharide (formation of which is inhibited by tunicamycin) is mandatory for the expression of lipoprotein lipase activity; lipoprotein lipase is active also in a high mannose form; and terminal glycosylation and oligosaccharide processing, which is inhibited by monensin, may be important for the appearance of heparin-releasable lipoprotein lipase and secretion of lipoprotein lipase into the medium.     

Interfacial reaction dynamics and acyl-enzyme mechanism for lipoprotein lipase-catalyzed hydrolysis of lipid p-nitrophenyl esters

The fatty acyl (lipid) p-nitrophenyl esters p-nitrophenyl caprylate, p-nitrophenyl laurate and p-nitrophenyl palmitate that are incorporated at a few mol % into mixed micelles with Triton X-100 are substrates for bovine milk lipoprotein lipase. When the concentration of components of the mixed micelles is approximately equal to or greater than the critical micelle concentration, time courses for lipoprotein lipase-catalyzed hydrolysis of the esters are described by the integrated form of the Michaelis-Menten equation. Least square fitting to the integrated equation therefore allows calculation of the interfacial kinetic parameters Km and Vmax from single runs. The computational methodology used to determine the interfacial kinetic parameters is described in this paper and is used to determine the intrinsic substrate fatty acyl specificity of lipoprotein lipase catalysis, which is reflected in the magnitude of kcat/Km and kcat. The results for interfacial lipoprotein lipase catalysis, along with previously determined kinetic parameters for the water-soluble esters p-nitrophenyl acetate and p-nitrophenyl butyrate, indicate that lipoprotein lipase has highest specificity for the substrates that have fatty acyl chains of intermediate length (i.e. p-nitrophenyl butyrate and p-nitrophenyl caprylate). The fatty acid products do not cause product inhibition during lipoprotein lipase-catalyzed hydrolysis of lipid p-nitrophenyl esters that are contained in Triton X-100 micelles. The effects of the nucleophiles hydroxylamine, hydrazine, and ethylenediamine on Km and Vmax for lipoprotein lipase catalyzed hydrolysis of p-nitrophenyl laurate are consistent with trapping of a lauryl-lipoprotein lipase intermediate. This mechanism is confirmed by analysis of the product lauryl hydroxamate when hydroxylamine is the nucleophile. Hence, lipoprotein lipase-catalyzed hydrolysis of lipid p-nitrophenyl esters that are contained in Triton X-100 micelles occurs via an interfacial acyl-lipoprotein lipase mechanism that is rate-limited by hydrolysis of the acyl-enzyme intermediate.     

Lipoprotein lipase: evidence for high- and low-affinity enzyme sites.

The kinetic constants for membrane-supported lipoprotein lipase have been determined for the enzyme active in lipoprotein triglyceride catabolism in perfused heart and adipose tissues, using a nonrecirculating system. Heart endothelial lipoprotein lipase reacted as a single population of high-affinity substrate binding sites (Km' 0.07 mM triglyceride). Km' (apparent Michaelis constant for the supported enzyme species) was independent of flow rate and the enzyme was rapidly released by heparin, suggestive of a superficial membrane binding site. Lipoprotein lipase active in perfused adipose tissue had significantly different kinetic properties, including a low substrate affinity (Km' 0.70 mM triglyceride), diffusion dependence of Km' at low flow rates, and slow release of enzyme by heparin. Adipose tissue may contain a small proportion of high affinity sites. While only a small proportion of total heart tissue lipoprotein lipase was directly active in triglyceride hydrolysis, this study suggests that the major part of lipoprotein lipase in adipose tissue may be involved in the hydrolysis of circulating lipoprotein triglyceride.     

Inspection of an Acyl-Enzyme Intermediate in a Lipase Reaction by Gas Chromatography-Mass Spectrometry and Modelling of the Reaction Mechanism

An acyl-enzyme intermediate proposed in the reaction mechanism of lipase was inspected by the exchange of oxygen between substrate (oleic acid) and solvent (¹⁸O-labelled water). Gas chromatography-mass spectrometry analysis supported the formation of an acyl-enzyme intermediate in the reaction mechanism through the observed incorporation of ¹⁸O into oleic acid. The incorporation did not occur in the absence of the lipase. When Ser residues were modified with diisopropylfluorophosphate, the activity of lipase OF 360 was markedly decreased. Photooxidation of His residues also resulted in a decrease in the activity of the lipase. Chemical modification studies suggested the existence of a charge relay system (Ser-His-Asp/Glu) in the active site. Based on these results, a model of the active site and reaction mechanism of the lipase are presented.     

Mode of action of tetrahydrolipstatin: a derivative of the naturally occurring lipase inhibitor lipstatin

Tetrahydrolipstatin is a specific lipase inhibitor derived from lipstatin, a lipid produced by Streptomyces toxytricini. In addition to pancreatic lipase, it is shown in the present study that tetrahydrolipstatin also inhibits human gastric lipase, carboxyl ester lipase (cholesterol esterase) of pancreatic origin and the closely related bile-salt-stimulated lipase of human milk. It does not inhibit the exocellular lipase from Rhizopus arrhizus or a lipase recently isolated from Staphylococcus aureus. In the presence of a water-insoluble substrate, such as tributyrin, the inhibition has the characteristics of an irreversible inactivation of the uncompetitive type, thus indicating that an enzyme.substrate.inhibitor complex is formed, which cannot undergo further reaction to yield the normal product. This reaction probably takes place at the aqueous/oil interface of the substrate. In aqueous solution, in the absence of substrate, the inhibition of carboxyl ester lipase by tetrahydrolipstatin has the characteristics of being reversible, and finally becomes of a temporary nature analogues to the trypsin-trypsin inhibitor system. It is suggested that an enzyme-inhibitor complex of an acyl-enzyme type is formed that is slowly hydrolysed, with water as the final acceptor, leaving an intact enzyme and an inactive form of the inhibitor. The enzyme thus consumes the inhibitor, which undergoes a chemical conversion, as indicated by a change in mobility in an appropriate thin-layer chromatographic system, indicating an increase in hydrophilicity. Evidence is presented that the reaction product is an acid and that the functional group of tetrahydrolipstatin is the beta-lactone reacting with the active site of the enzyme.     

Lipoprotein lipase is expressed by glomerular mesangial cells

Lipoprotein lipase expressed by the vasculature plays a key role in atherogenesis by enhancing the binding and uptake of lipoproteins and, thereby, leading to the formation of lipid-loaded foam cells. Hyperlipidemia also accelerates the progression of glomerular diseases and addition of exogenous lipoprotein lipase to mesangial cells has been shown to lead to an enhanced binding of lipoproteins to these cells. Despite such potential importance, the expression of endogenous lipoprotein lipase by cells of the glomeruli has, as yet, not been investigated. We show here for the first time that mesangial cells, but not epithelial cells, express lipoprotein lipase. The minimal lipoprotein lipase gene promoter was active in mesangial cells and inhibited by interferon-gamma, which is known to suppress its expression.     

Lipoprotein lipase. Mechanism of formation of triglyceride-rich remnant particles from very low density lipoproteins and chylomicrons.

The catalytic rate of membrane-supported lipoprotein lipase has been determined for chylomicron and very low density lipoprotein substrates during the formation of triglyceride-depleted ("remnant") particles. Both lipoprotein species and their generated remnant products were competitive substrates for lipase activity. Remnant formation from each species was associated with decreasing kc but an unchanged apparent Km. This finding was confirmed from the rate of plot of total triglyceride catabolism by lipase at low substrate concentrations. When compared with the major very low density lipoprotein fraction (Sf 100-400), a fraction isolated from plasma with a lower flotation rate (Sf 40-100) had a lipid composition and decreased kc compatible with this representing a physiological remnant particle.     

Phenyl-n-butylborinic acid is a potent transition state analog inhibitor of lipolytic enzymes

The cholesterol esterase and lipoprotein lipase catalyzed hydrolyses of the water-soluble substrate p-nitrophenyl butyrate are competitively inhibited by butaneboronic acid and phenylboronic acid. Phenyl-n-butylborinic acid has been synthesized and characterized as an ultrapotent transition state analog inhibitor: Ki = 2.9 +/- 0.6 nM and 1.7 +/- 0.3 microM for the cholesterol esterase and lipoprotein lipase reactions, respectively. These results are interpreted in terms of transition state structure and stabilization.     

Hydrolysis of chylomicron polyenoic fatty acid esters with lipoprotein lipase and hepatic lipase

The lipolysis of rat chylomicron polyenoic fatty acid esters with bovine milk lipoprotein lipase and human hepatic lipase was examined in vitro. Chylomicrons obtained after feeding fish oil or soy bean oil emulsions were used as substrates. The lipolysis was followed by gas chromatography or by using chylomicrons containing radioactive fatty acids. Lipoprotein lipase hydrolyzed eicosapentaenoic (20:5) and arachidonic acid (20:4) esters at a slower rate than the C14-C18 acid esters. More 20:5 and 20:4 thus accumulated in remaining tri- and diacylglycerols. Eicosatrienoic, docosatrienoic and docosahexanoic acids exhibited an intermediate lipolysis pattern. When added together with lipoprotein lipase, hepatic lipase increased the rate of lipolysis of 20:5 and 20:4 esters of both tri- and diacylglycerols. Addition of NaCl (final concentration 1 M) during the course of lipolysis inhibited lipoprotein lipase as well as the enhancing effect of hepatic lipase on triacylglycerol lipolysis. Hepatic lipase however, hydrolyzed diacylglycerol that had already been formed. Chylomicron 20:4 and 20:5 esters thus exhibit a relative resistance to lipoprotein lipase. It is suggested that the tri- and diacylglycerol species containing these fatty acids may accumulate at the surface of the remnant particles and act as substrate for hepatic lipase during a concerted action of this enzyme and lipoprotein lipase.     

Immunological studies on bovine milk lipoprotein lipase. Effects of Fab fragments on enzyme activity

Rabbit antiserum was prepared against purified bovine mild lipoprotein lipase. Immunoelectrophoresis of lipoprotein lipase gave a single precipitin line against the antibody which was coincident with enzyme activity. The gamma-globulin fraction inhibited heparin-releasable lipoprotein lipase activity of bovine arterial intima, heart muscle and adipose tissue. The antibody also inhibited the lipoprotein lipase activity from adipose tissue of human and pig, but not that of rat and dog. Fab fragments were prepared by papain digestion of the gamma-globulin fraction. Fab fragments inhibited the lipoprotein lipase-catalyzed hydrolysis of dimyristoylphosphatidylcholine vesicles and trioleoylglycerol emulsions to the same extent. The Fab fragments also inhibited the lipolysis of human plasma very low density lipoproteins. The change of the kinetic parameters for the lipoprotein lipase-catalyzed hydrolysis of trioleoylglycerol by the Fab fragments was accompanied with a 3-fold increase in Km and a 10-fold decrease in Vmax. Preincubation of lipoprotein lipase with apolipoprotein C-II, the activator protein for lipoprotein lipase, did not prevent inhibition of enzyme activity by the Fab fragments. However, preincubation with dipalmitoylphosphatidylcholine-emulsified trioleoylglycerol or Triton X-100-emulsified trioleoylglycerol had a protective effect (remaining activity 7.0 or 25.8%, respectively, compared to 1.0 or 0.4% with no preincubation). The addition of both apolipoprotein C-II and substrate prior to the incubation with the Fab fragments was associated with an increased protective effect against inhibition of enzyme activity; remaining activity with dipalmitoylphosphatidylcholine-emulsified trioleoylglycerol was 40.6% and with Triton X-100-emulsified trioleoylglycerol, 45.4%. Human plasma very low density lipoproteins also protected against the inhibition of enzyme activity by the Fab fragments. These immunological studies suggest that the interaction of lipoprotein lipase with apolipoprotein C-II in the presence of lipids is associated with a conformational change in the structure of the enzyme such that the Fab fragments are less inhibitory. The consequence of a conformational change in lipoprotein lipase may be to facilitate the formation of an enzyme-triacylglycerol complex so as to enhance the rate of the lipoprotein lipase-catalyzed turnover of substrate to products.     

Rational design of alpha-keto triglyceride analogues as inhibitors for Staphylococcus hyicus lipase.

We have synthesized a series of alpha-keto triglyceride analogues as inhibitors for the lipase from Staphylococcus hyicus (SHL). Hydrolysis at positions 1 and 2 was prevented by replacement of the ester bonds by nonhydrolyzable ether, carbamoyl, or amide bonds, and an alpha-keto fatty acid was introduced at position 3. Such compounds act as competitive inhibitors of SHL. Inhibition must be caused by the presence of the alpha-keto functions, since the compounds containing an ester or a hydroxyl group in position 3 did not inhibit the enzyme. We propose that our inhibitors react with the active site Ser of the lipase, hereby mimicking the tetrahedral intermediate occurring in substrate hydrolysis. We conclude that the localization of the alpha-keto triglycerides is very important for inhibition because only those compounds which are insoluble in water are lipase inhibitors. In addition, other specific protein-inhibitor interactions, probably with the carbonyl oxygen at position 1 and/or 2, improve inhibitor binding. This makes the compounds with amide or carbamoyl groups at positions 1 and 2 better inhibitors than their ether counterparts. The inhibitory power could be improved further by replacing the oxygen at position 3 by an amido group. The resulting inhibitor 1, 2-diethylcarbamoyl-3-amido-alpha-ketododecanoylglycerol has a Ki value of 0.008 mol %, indicating that it binds approximately 125-fold tighter than the substrate. These results illustrate that effective lipase inhibitors can be designed by combining an alpha-keto group with good micellar solubility and optimal protein-inhibitor interactions.     

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

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

The effect in vitro of high-density lipoprotein on hydrolysis of triacylglycerol by lipoprotein lipase.

In an incubation system in vitro with fully activated Intralipid as substrate, rat high-density lipoprotein inhibits the hydrolysis of triacylglycerol by lipoprotein lipase from rat adipose tissue, but does not inhibit hydrolysis by the enzyme from bovine milk. The pattern of inhibition suggests that substrate and high-density lipoprotein may compete for association with rat adipose-tissue lipoprotein lipase.     

Inhibition of human and rat lipoprotein lipase by high-density lipoprotein

The hydrolysis in vitro of preactivated Intralipid (an artificial triacylglycerol-phospholipid emulsion) by rat adipose tissue lipoprotein lipase is inhibited by rat high-density lipoprotein (HDL). The aim of this work was to investigate whether human lipoprotein lipase was also inhibited, the mechanism of inhibition of the rat enzyme by HDL, and the role of the various individual apolipoproteins. Both human and rat lipoprotein lipase from post-heparin plasma are inhibited by HDL. This inhibition is considerably decreased if the HDL is first made 'apolipoprotein poor' by removal of some transferable apolipoproteins. In contrast, both native and apolipoprotein poor HDL inhibit the hydrolysis of Intralipid by rat hepatic lipase. Apolipoproteins C and E, either free in solution or attached to lipid vesicles, inhibit the hydrolysis of activated Intralipid by rat lipoprotein lipase to a maximum of 85% and 50%, respectively. Apolipoprotein A attached to vesicles gives little inhibition. HDL apolipoprotein and apolipoprotein C compete with the substrate for binding to lipoprotein lipase with apolipoprotein C having a higher affinity for the enzyme than HDL apolipoprotein. The inhibition of lipoprotein lipase by HDL can be explained by the association of the constituent apolipoproteins, in particular apolipoprotein C, with the enzyme so that there is less enzyme available to act on substrate.     

CL-proteins and the regulation of lipoprotein lipase activity in locust flight muscle

Lipoprotein lipases in the flight muscles of Locusta migratoria show a marked substrate specificity: diacylglycerols associated with the adipokinetic hormone (AKH)-induced lipoprotein, A+, are hydrolysed at 4 to 5 times the rate of those associated with the lipoprotein in resting (non-hormone-stimulated) locusts, Ayellow. To determine the basis for this discrimination, the effect on the activity of flight muscle lipoprotein lipase of CL-proteins, a major constituent of lipoprotein A+, but not of Ayellow, has been investigated; they inhibit the flight muscle enzyme in a competitive manner whether activity is measured with a natural lipoprotein substrate, a lipid emulsion or a water soluble substrate. Experiments in vivo suggest that the flight muscle enzyme is normally inhibited in resting (non-AKH-stimulated) locusts but, interestingly, injection of synthetic AKH-I relieves the inhibition and increases the activity by 30 to 40%. This is not a direct effect of the hormone on the enzyme, but appears to be related to the hormone-induced formation of lipoprotein A+, so that the majority of CL-proteins in the haemolymph become bound to this lipoprotein and the concentration of free CL-proteins is markedly reduced. We suggest that CL-proteins play a major role in the regulation of lipoprotein lipase in locust flight muscle.     

The lipase C-terminal domain. A novel unusual inhibitor of pancreatic lipase activity

In vertebrates, dietary fat digestion mainly results from the combined effect of pancreatic lipase, colipase, and bile. It has been proposed that in vivo lipase adsorption on oil-water emulsion is mediated by a preformed lipase-colipase-mixed micelle complex. The main lipase-colipase binding site is located on the C-terminal domain of the enzyme. We report here that in vitro the isolated C-terminal domain behaves as a potent noncovalent inhibitor of lipase and that the inhibitory effect is triggered by the presence of micelles. Lipase inhibition results from the formation of a nonproductive C-terminal domain-colipase-micelle ternary complex, which competes for colipase with the active lipase-colipase-micelle ternary complex, thus diverting colipase from its lipase-anchoring function. The formation of such a complex has been evidenced by molecular sieving experiments. This nonproductive complex lowers the amount of active lipase thus reducing lipolysis. Preliminary experiments performed in rats show that the C-terminal domain also behaves as an inhibitor in vivo and thus could be considered a potential new tool for specifically reducing intestinal lipolysis.     

Gastric lipase: crystal structure and activity

Fat digestion in humans requires not only the classical pancreatic lipase but also gastric lipase, which is stable and active despite the highly acidic stomach environment. We have solved the structure of recombinant human gastric lipase at 3.0 A resolution, the first structure to be described within the mammalian acid lipase family. This globular enzyme (379 residues) consists of a core domain, belonging to the alpha/beta hydrolase fold family, and an extrusion domain. It possesses a classical catalytic triad (Ser 153, His 353, Asp 324) and an oxyanion hole (NH groups of Gln 154 and Leu 67). Four N-glycosylation sites were identified on the electron density maps. The catalytic serine is deeply buried under the extrusion domain, which is composed of a 'cap' domain and a segment consisting of 30 residues, which can be defined as a lid. Its displacement is necessary for the substrates to access the active site. A phosphonate inhibitor was positioned in the active site which clearly suggests the location of the hydrophobic substrate binding site.     

Spectral observation of an acyl-enzyme intermediate of lipoprotein lipase

There have been several studies indicating that hydrolysis reactions of fatty acid esters catalyzed by lipases proceed through an acyl-enzyme intermediate typical of serine proteases. In particular, one careful kinetic study with the physiologically important enzyme lipoprotein lipase (LPL) is consistent with rate-limiting deacylation of such an intermediate. To observe the spectrum of acyl-enzyme and study the mechanism of LPL-catalyzed hydrolysis of substrate, we have used a variety of furylacryloyl substrates including 1,2-dipalmitoyl-3-[(beta-2-furylacryloyl)triacyl]glyceride (DPFATG) to study the intermediates formed during the hydrolysis reaction catalyzed by the enzyme. After isolation and characterization of the molecular weight of adipose LPL, we determined its extinction coefficient at 280 nm to quantitate the formation of any acyl-enzyme intermediate formed during substrate hydrolysis. We observed an intermediate at low pH during the enzyme-catalyzed hydrolysis of (furylacryloyl)imidazole. This intermediate builds early in the reaction when a substantial amount of substrate has hydrolyzed but no product, furylacrylate, has been formed. The acyl-enzyme has a lambda max = 305 nm and a molar extinction coefficient of 22,600 M-1 cm-1; these parameters are similar to those for furylacryloyl esters including the serine ester. These data provide the first spectral evidence for a serine acyl-enzyme in lipase-catalyzed reactions. The LPL hydrolysis reaction is base catalyzed, exhibiting two pKa values; the more acidic of these is 6.5, consistent with base catalysis by histidine. The biphasic rates for substrate disappearance or product appearance and the absence of leaving group effect indicate that deacylation of intermediate is rate limiting.     

Kinetics and phospholipid specificity of apolipoprotein N-acyltransferase

The enzyme apolipoprotein N-acyltransferase (Lnt) is an integral membrane protein that catalyzes the last step in the post-translational modification of bacterial lipoproteins. Lnt undergoes covalent modification in the presence of phospholipids resulting in a thioester acyl-enzyme intermediate. It then transfers the acyl chain to the α-amino group of the N-terminal diacylglyceryl-modified cysteine of apolipoprotein, leading to the formation of mature triacylated lipoprotein. To gain insight into the catalytic mechanism of this two-step reaction, we overproduced and purified the enzyme of Escherichia coli and studied its N-acyltransferase activity using a novel in vitro assay. The purified enzyme was fully active, as judged by its ability to form a stable thioester acyl-enzyme intermediate and N-acylate the apo-form of the murein lipoprotein Lpp in vitro. Incorporation of [(3)H]palmitate and mass spectrometry analysis demonstrated that Lnt recognized the synthetic diacylglyceryl-modified lipopeptide FSL-1 as a substrate in a mixed micelle assay. Kinetics of Lnt using phosphatidylethanolamine as an acyl donor and FSL-1 as a substrate were consistent with a ping-pong type mechanism, demonstrating slow acyl-enzyme intermediate formation and rapid N-acyl transfer to the apolipopeptide in vitro. In contrast to earlier in vitro observations, the N-acyltransferase activity was strongly affected by the phospholipid headgroup and acyl chain composition.