Essential histidine residues in lysolecithin:lysolecithin acetyltransferase from rabbit lung

Both activities of rabbit lung lysolecithin:lysolecithin acyltransferase (EC 3.1.1.5), hydrolysis and transacylation, are inactivated by diethylpyrocarbonate. The reaction follows pseudo-first-order kinetics, and second-order rate constants of 1.17 mM-1min-1 for hydrolysis and 0.56 mM-1 min-1 for transacylation were obtained at pH 6.5 and 37 degrees C. The rate of inactivation is dependent on pH, showing the involvement of a group with a pK of 6.5. The difference spectra showed an increase in absorbance at 242 nm, indicating the modification of histidine residues. The activity lost by diethylpyrocarbonate modification can be partially recovered by hydroxylamine treatment. The statistical analysis of residual fractional activity versus the number of modified histidine residues leads to the conclusion that two histidine residues are essential for the hydrolytic activity, whereas transacylation activity depends on only one essential histidine. The substrate and substrate analogs protected the enzyme against inactivation by diethylpyrocarbonate, suggesting that the essential residues are located at or near the active site of the enzyme.     

The kinetic mechanism of acyl-CoA:lysolecithin acyltransferase from rabbit lung

Acyl-CoA:lysolecithin acyltransferase is a key enzyme in the deacylation-reacylation pathway of biosynthesis of molecular species of lecithin. However, the mechanism of the reaction has been little studied. In this paper, the kinetic mechanism of acyl-CoA:lysolecithin acyltransferase, partially purified from rabbit lung, is studied. The double-reciprocal plots of initial velocity vs substrate concentration gave two sets of parallel lines which fitted to a ping-pong equation with the following parameters: Km (palmitoyl-CoA) = 8.5 +/- 2 microM, Km (lysolecithin) = 61 +/- 16 microM, and V = 18 +/- 4 nmol/min/mg protein. Inhibition studies by substrates, alternate substrates, and products supported the ping-pong mechanism, although some nonclassical behavior was observed. Palmitoyl-CoA did not inhibit even at concentrations of 100 Km. In contrast, lysolecithin was a dead-end inhibitor with a dissociation constant of Ki = 930 +/- 40 microM. Alternate substrates and CoA showed alternate pathways for the reaction due to the formation of ternary complexes. Dipalmitoylphosphatidylcholine inhibition pointed to an isomerization of the free enzyme prior to the start of the reaction. From these results, an iso-ping-pong kinetic mechanism for lysolecithin acyltransferase is proposed. The kinetic steps of the reaction are correlated with previous chemical studies of the enzyme.     

Essential residues in lysolecithin:lysolecithin acyltransferase from rabbit lung: assessment by chemical modification

The inhibition of lysolecithin:lysolecithin acyltransferase by several specific reagents was studied. Diisopropyl fluorophosphate (DFP) completely inhibited both activities at a concentration of 4 mM. Activity was not protected by substrate and the enzyme showed a change in circular dichroism spectrum upon treatment with inhibitor. Phenylmethanesulfonyl fluoride, another serine-specific reagent, did not inhibit either hydrolysis or transacylation. Therefore, we suggest that DFP does not modify an active serine in the catalytic site. p-Hydroxymercury benzoate and N-ethylmaleimide (NEM) abolished both activities of the enzyme. The presence of substrate partially protected against inactivation. Far-uv CD spectrum of NEM-modified enzyme revealed no changes in protein structure. The existence of two classes of essential cysteine residues was deduced from kinetics of NEM inactivation. Both classes differ in NEM reactivity and also in their participation in the catalytic mechanism. A tyrosine-specific reagent, tetranitromethane, also inhibited hydrolysis and transacylation, following first-order kinetics. The partial protection by substrate suggested the possible existence of essential tyrosines near the active site. At pH 5.0 N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline inactivated hydrolysis but not transacylation. However, both of them remained unchanged at pH 6.5. The substrate prevented the loss of hydrolytic ability. Therefore, a carboxyl residue participating just in the catalytic mechanism of hydrolysis is proposed.     

Effect of albumin on acyl-CoA: lysolecithin acyltransferase,lysolecithin : lysolecithin acyltransferase and acyl-CoA hydrolase from rabbit lung

Acyl-CoA : lysolecithin and lysolecithin : lysolecithin acyltransferases, as well as acyl-CoA hydrolase are important enzymes in lung lipid metabolism. They use amphiphylic lipids as substrates and differ in subcellular localization. In this sense, lipid-protein interactions can be an essential factor in their activity. We have studied the effect of albumin, as lipid-binding protein model, in the activities of these enzymes. Acyl-CoA hydrolase was inhibited in the presence of albumin, whereas acyl-CoA : lysolecithin acyltransferase showed a complex effect of activation depending on both albumin concentration and palmitoyl-CoA/lysolecithin molar ratio. Lysolecithin : lysolecithin acyltransferase was affected differentially on its two activities. Hydrolysis remained unaffected and transacylation was inhibited by albumin. These results are consequence of the interaction of albumin with both lipidic substrates that changes their critical micellar concentration.Abbreviations TNS 6-(p-toluidino)-2-naphthalene-sulfonic acid - CMC Critical Micellar Concentration - LP Lysolecithin (1-acyl-sn-glycero-3-phosphocholine) - PalmCoA palmitoyl-CoA     

Effect of lipids on activity and conformation of lysolecithin:lysolecithin acyltransferase from rabbit lung

Summary Lysolecithin:lysolecithin acyltranferase is an enzyme which in several previous studies has shown a dual behavior catalyzing two types of reaction, transacylation or hydrolysis, with the same substrate. Both activities have shown to be dependent on several environmental conditions and among them, the presence of lipids.The addition of several classes of lipids activated in all the cases the enzyme, decreasing the hydrolysis/transacylation molar ratio. This effect was higher for PC/PE/Chol mixture than for other lipids assayed. Circular dichroism spectra of the enzyme did not show any change with the addition of lipids, concluding that the effect of lipids was not due to any structural change in the protein. The hypothesis has been made of an influence of lipids on the physical state of the substrate as well as, possibly, on the enzyme-substrate interaction.The significance of these effects on the physiological role of lysolecithin:lysolecithin acyltransferase from soluble fraction of rabbit lung is discussed.Abbreviations Chol cholesterol - CMC critical micellar concentration - DPPC dipalmitoylphosphatidylcholine - FA fatty acid - H/T hydrolysis/transacylation molar ratio - LPC lysophosphatidylcholine - PC phosphatidylcholine - PE phosphatidylethanolamine - TG triglyceride - UV ultraviolet     

Demonstration of enzymatic conversion of lysolecithin to lecithin in normal human plasma

An enzyme in normal human plasma that converts [1-acyl ¹⁴C] lysolecithin to lecithin is demonstrated. This enzyme is inhibited by heparin and is not derived from platelets or other blood elements. The synthesis of lecithin from labeled lysolecithin was not stimulated by ATP and CoA or by oleyl CoA and there was nearly an equal distribution of labeled fatty acid between the two positions of lecithin indicating that the enzyme may be a lysolecithin: lysolecithin acyl transferase (LLAT). The enzyme is associated with the lipoproteins of the plasma, and may have a physiological role in the formation of saturated cholesterol esters in plasma.     

Spontaneous vesiculation of uncharged phospholipid dispersions consisting of lecithin and lysolecithin

The work presented here demonstrates that the phenomenon of spontaneous vesiculation is not restricted to charged lipids and lipid mixtures, but occurs also in isoelectric phospholipid mixtures consisting of egg phosphatidylcholine (EPC) and egg lysophosphatidylcholine (lyso-EPC). 1H high-resolution NMR and freeze-fracture electron microscopy have been used to characterize the mixed EPC/lyso EPC dispersions in excess H2O. The predominant phase in these mixed phospholipid dispersions is smectic (lamellar) at least up to approximately 70% lysophosphatidylcholine. The type of phospholipid aggregate formed in excess H2O depends on the mole ratio diacyl to monoacyl phosphatidylcholine. The dispersive (lytic) action of lysophosphatidylcholine on phosphatidylcholine bilayers becomes effective at lysophospholipid contents in excess of approximately 10%. Large multilamellar liposomes are disrupted and replaced by smaller particles, mainly unilamellar vesicles. Between 30 and 70% lysophosphatidylcholine a significant proportion of the total phospholipid is present as small unilamellar vesicles (SUV) of a diameter of 23 nm (range: 20-70 nm). At even higher lysophosphatidylcholine contents the fraction of phospholipid present as small mixed micelles with a diameter smaller than about 14 nm grows at the expense of the vesicular structures. There is a second effect of increasing the quantity of lysophosphatidylcholine in phosphatidylcholine bilayers: the presence of lysophosphatidylcholine in excess of 10% renders the phospholipid bilayer more permeable to ions as compared to pure phosphatidylcholine bilayers. The key factor in inducing spontaneous vesiculation is probably not the charge but the wedge-like shape of the lysophospholipid molecule. The molecular shape may give rise to an asymmetric distribution of lysophosphatidylcholine between the two halves of the bilayer, thus stabilizing highly curved bilayers as present in SUV.     

Activity of cholinephosphotransferase, lysolecithin: lysolecithin acyltransferase and lysolecithin acyltransferase in the developing mouse lung.

1. The present study presents the activity profiles of cholinephosphotransferase, lysolecithin:lysolecithin acyltransferase and lysolecithin acyltransferase at different stages of development of the mouse lung. 2. The specific activity of cholinephosphotransferase, a key enzyme in the de novo synthesis of phosphatidylcholine, increases during the later stages of fetal development until it reaches a maximal value at a gestational age of 17 days, i.e. 2 days before term. Thereafter, the activity of the enzyme declines again until around term. 2. The specific activity of lysolecithin:lysolecithin acyltransferase which catalyzes the transesterification between two molecules of 1-acyl-sn-glycero-3-phosphocholine, appears to be much lower than that of cholinephosphotransferase at gestational ages below 18 days. However, around day 18, the specific activity of lysolecithin:lysolecithin acyltransferase increases dramatically until it almost equals the maximal activity of cholinephosphotransferase measured on day 17. 4. The specific activity of lysolecithin acyltransferase, which catalyzes the direct acylation of 1-acyl-sn-glycero-3-phosphocholine, does not change significantly during the prenatal development and is lower than that of either lysolecithin:lysolecithin acyltransferase or cholinephosphotransferase at all stages of development. 5. These results are discussed in view of the possible role of these enzymes in the biosynthesis of pulmonary 1,2-dipalmitoyl-sn-glycero-3-phosphocholine.     

Differential scanning calorimetry on mixtures of lecithin,lysolecithin and cholesterol

The effect of increasing concentrations of lysolecithin (1-palmitoyl-sn-glycerol-3-phosphorylcholine) on the gel → liquid crystal thermal transition of lecithin (1,2-dipalmitoyl-sn-glycerol-3-phosphorylcholine) in the aqueous phase was studied by differential scanning calorimetry (DSC). Lysolecithin showed an endothermic transition at 3.4°C whereas the transition of the lecithin occurred at 42°C. No phase separation could be observed calorimetrically at lysolecithin concentrations up to 60 mol%. Freeze etch electron microscopy showed that mixtures containing as much as 50 mol% lysolecithin exist in a lamellar phase. The lysolecithin was found to cause an initial slight increase in the enthalpy of transition followed by a gradual decrease. The enthalpy increased again at very high lysolecithin concentrations. The lysolecithin also caused a non-linear decrease in the temperature at which the lecithin transition took place.Cholesterol was found to decrease the enthalpy of transition of the lysolecithin, eliminating it at a concentration of 50 mol%. Cholesterol caused an increase in the temperature at which the lysolecithin transition took place.     

Substrate selectivity of acyl-CoA:lysolecithin acyltransferase from rabbit lung

The influence of both polar head and acyl chain of lysophospholipid on the activity of partially purified acyl-CoA:lysolecithin acyltransferase from rabbit lung was studied. It was concluded that the presence of methyl groups on the nitrogen of the base was essential for recognition of lysophospholipid as substrate by the enzyme. With respect to the acyl chain length and saturation, the activity followed the order: 16:0 approximately equal to 18:1 greater than 14:0 greater than greater than greater than 18:0 approximately equal to 12:0. Also, the effect on the activity of the acyl chain on acyl-CoA was studied. The activity showed great selectivity for saturated acyl-CoAs. The activity with polyunsaturated fatty acids was very low and in the case of arachidonoyl-CoA was almost negligible. The comparison between crude microsomal preparations and partially purified preparations allowed to suggest that it could exist two different acyl-CoA:lysolecithin acyltransferases differing in their selectivity towards saturated and unsaturated fatty acids.     

Interaction of lysolecithin micelles and lecithin vesicles with apolipoprotein Gln I from serum high density lipoproteins

Lysolecithin and apoLP-Gln I, mixed without sonication and analysed ultracentrifugally, formed a complex of density 1.063–1.21 g/ml. Formation of this lysolecithin-apolipoprotein complex was accompanied by changes in apolipoprotein conformation and fluorescence. ApoLP-Gln I, when incubated with lecithin vesicles, primarily formed complexes of density < 1.063 g/ml and formed few complexes of density 1.063–1.21 g/ml. Preliminary studies on mixtures containing apoLP-Gln I, lysolecithin, and lecithin vesicles are also described and suggest that apoLP-Gln I in a 1.063–1.21 g/ml complex can bind up to 200 molecules of phospholipid per molecule of apolipoprotein.     

Evidence of a pH-dependent conformational change at the active site of lysolecithin:lysolecithin acyltransferase from rabbit lung

It has been shown that both activities, hydrolysis and transacylation, of lysolecithin:lysolecithin acyltransferase, as well as the conformation of the polypeptide are critically dependent on a pK around 5.8, but the question remains if the same residue(s) is responsible for the conformational change and the loss of activity. In this paper, ultrasonic cavitation is used to study the pH-dependent inactivation. The results show that there are two first-order inactivation constants which depend on pH and that the transition between them has a pK of 5.9. As the constants of ultrasonic inactivation are very dependent on the accessibility of the residues it is concluded that the conformational change modifies the accessibility of the active site.     

Lysolecithin:lysolecithin acyltransferase from rabbit lung. A conformational study

The enzyme lysolecithin:lysolecithin acyltransferase from rabbit lung has been found to have a relatively disordered conformation in solutions of high ionic strength. The protein exhibited an ordering of structure when salt was suppressed. This conformational change was concomitant with the loss of transacylase activity, the hydrolytic reaction remaining unchanged. Addition of NaCl caused a progressive disordering of structure with a parallel increase of transacylase activity. The acid denaturation of the protein, at low and high ionic strengths, showed that the ionization of groups with pK in the range 5.9-6.4 was essential for denaturation. The structure was stable at basic pH. The addition of lipids resulted in a non-specific stabilization of the disordered conformation, in the same manner as the addition of NaCl. From these results, it is suggested that there are two conformations for this protein which differ in their ability to bind lysolecithin molecules in the enzyme deacylation step of the reaction. This hypothesis agrees with previously published properties of the enzyme, concerning aggregation with other proteins and kinetic data. From the amino acid composition and conformational properties, the authors suggest that this enzyme could be a peripheral membrane protein.