Secondary Ester Bond Research Articles

Xanthophyll esters are hydrolysed in the presence of recombinant human pancreatic lipase

Fruit-derived bioactive xanthophyll esters have to be cleaved in the human gastrointestinal tract before absorption of free xanthophylls is possible. One candidate for ester hydrolysis is human pancreatic lipase. For estimation of their activity, an in vitro assay using the recombinant enzyme human pancreatic lipase (rHPL) and porcine colipase was used. Extracts of fruits were incubated with rHPL/colipase for 21 h at 37 °C. Activity of rHPL was demonstrated by an increase of free xanthophylls formed during the incubation. An extremely low activity was detected with all substrates. HPLC–(APcI)MS studies proved that lutein diesters were preferentially cleaved at the β-ionone ring. This is the first report which shows that xanthophyll esters can be cleaved in the presence of rHPL, suggesting either an unexpected secondary ester bond hydrolysis occurring within rHPL active site or, more probably, a reaction induced by other amino acids of HPL such as observed earlier with p-nitrophenyl acetate.

Substrate specificity and effects of water-miscible solvents on the activity and stability of extracellular lipase from Streptomyces rimosus

Substrate specificity, regioselectivity and transesterification activity of purified extracellular lipase from Streptomyces rimosus were investigated. The enzyme showed pronounced lipolytic activity toward a number of triacylglycerols and oils of vegetable and animal origin. It hydrolyzed most efficiently medium chain length fatty acid glycerol esters (C8–C12). There was preference for the esters of C16 and C18 unsaturated fatty acids over C16 and C18 saturated fatty acid esters, as well as for triacylglycerol substrate with cis double bond (triolein) versus trans double bond (trielaidin). Streptomyces rimosus lipase hydrolyzed primary and secondary ester bonds in triacylglycerols (triolein and 2,3-dimercapto-1-propanol tributyrate). The lipase catalyzed the hydrolysis of poly(oxyethylene) sorbitan monoesters (Tween 20–80) with rate comparable for that determined with triacylglycerols and oils. Several water-miscible solvents enhanced the lipase activity. 1,4-Dioxane activated the enzyme in a broad concentration range, up to 4-fold. Lipase was stable in solvent mixtures containing 50% (v/v) ethanol, 1,4-dioxane, acetonitrile or acetone. Tetrahydrofuran and N,N-dimethylformamide (both 50%) inactivated the enzyme with t 1/2 of 5 min and t 1/2 of 2 h, respectively. Transesterification of racemic 1-phenyl ethanol with vinyl acetate, catalyzed by extracellular lipase from Streptomyces rimosus in n-hexane, proceeded with partial R-enantioselectivity.

Stereochemical and positional specificity of the lipase/acyltransferase produced by Aeromonas hydrophila.

Aeromonas species secrete a glycerophospholipid-cholesterol acyltransferase (GCAT) which shares many properties with mammalian plasma lecithin-cholesterol acetyltransferase (LCAT). We have studied the stereochemical and positional specificity of GCAT against a variety of lipid substrates using NMR spectroscopy as well as other assay methods. The results show that both the primary and secondary acyl ester bonds of L-phosphatidylcholine can be hydrolyzed but only the sn-2 fatty acid can be transferred to cholesterol. The enzyme has an absolute requirement for the L configuration at the sn-2 position of phosphatidylcholine. The secondary ester bond of D-phosphatidylcholine cannot be hydrolyzed, and this lipid is not a substrate for acyl transfer. In contrast to the phospholipases, but similar to LCAT, the enzyme does not interact stereochemically with the phosphorus of phosphatidylcholine. In fact, the phosphorus is not required for enzyme activity, as GCAT will also hydrolyze monolayers of diglyceride, although at much lower rates.

A monolayer and bulk study on the kinetic behavior of Pseudomonas glumae lipase using synthetic pseudoglycerides

A heat-stable lipase from Pseudomonas glumae was purified to homogeneity. Its positional and stereospecific properties were investigated and compared with those of the well-known porcine pancreatic lipase. The kinetic properties of both enzymes were determined by use of six isomeric synthetic pseudoglycerides all composed of a single hydrolyzable fatty acyl ester bond and two lipase-resistant groups: one acylamino and one ether function. Two enzyme assay techniques were applied: a detergent-free system, the monomolecular surface film technique, and the pH-stat technique using clear micellar solutions of substrate in the presence of Triton X-100. Regarding the cleavage of primary ester bonds, P. glumae lipase possesses no stereopreference. In contrast, a large stereopreference in favor of the R-isomer is found for the hydrolysis of secondary ester bonds. Secondary ester bonds are efficiently cleaved by the lipase, which makes it of potential interest for enzymatic synthetic purposes. For the hydrolysis of this R-isomer a correlation between the experimental catalytic turnover rate and the binding constant for micelles was observed. The kinetic data of P. glumae lipase have been analyzed in terms of the scooting and hopping models for the action of lipolytic enzymes [Upreti, G.C., & Jain, M.K. (1980) J. Membr. Biol. 55, 113-121]. The results presented in this study are best explained by assuming that glumae lipase leaves the interface after a limited number of catalytic cycles.

Stereoselectivity of lipases. I. Hydrolysis of enantiomeric glyceride analogues by gastric and pancreatic lipases, a kinetic study using the monomolecular film technique

In the present study, porcine pancreatic lipase, rabbit gastric lipase, and human gastric lipase stereospecificity toward enantiomeric glyceride derivatives was kinetically investigated using the monomolecular film technique. Pseudoglycerides such as enantiomeric 1(3)-alkyl-2,3(1,2)-diacyl-sn-glycerol, enantiomeric 1(3)-alkyl-2-acyl-sn-glycerol, or enantiomeric 1(3)-acyl-2-acylamino-2-deoxy-sn-glycerol were synthesized in order to assess the lipase stereoselectivity during the hydrolysis of either the primary or the secondary ester position of these glycerides analogues. The cleaved acyl moiety was the same in both enantiomers, thereby excluding the possibility of effects occurring due to fatty acid specificity. We observed a porcine pancreatic lipase sn-3 stereoselectivity when using the enantiomeric 1(3)-alkyl-2-acylamino-2-deoxy-sn-glycerol (diglyceride analogue) which contrasted with the lack of stereoselectivity observed when using the enantiomeric 1(3)-alkyl-2,3(1,2)-diacyl-sn-glycerol (triglyceride analogue). The gastric lipases, in contrast to the pancreatic lipase, preferentially catalyze the hydrolysis of the primary sn-3 ester bond of the enantiomeric monoakyl-diacyl pair tested. From these kinetic data, high hydrolysis rates and no chiral discrimination were observed in the case of rabbit gastric lipase, whereas low rates and a clear chiral discrimination was noticed in the case of human gastric lipase during hydrolysis of the acyl chain from the secondary ester bond of 1(3)-alkyl-2-acyl enantiomers. It is particularly obvious that in the case of human gastric lipase decreasing the lipid packing increases the lipase sn-3 stereopreference during hydrolysis of the primary ester bond of the enantiomeric 2-acylamino derivatives (diglyceride analogue).

Diacylglycerol hydrolysis in rat liver lysosomes

The matrix of rat liver lysosomes exhibits high hydrolytic activity towards 1,2-diacylglycerol with an optimum at pH 4.0. The lipolytic reaction follows Michaelis-Menten kinetics (apparent V max 470 nmol hydrolysed min per mg protein; apparent K m71 μM 1,2-dioleoylglycerol). Formation of 1- and 2-monooleoylglycerols indicates an initial attack at both the primary and secondary ester bonds. The lysosomal matrix also catalyses (re)acylation reactions, i.e. the formation of 1,2-diacylglycerol from 2-monoacylglycerol and free fatty acid. However, (re)acylation proceeds at a far lower rate than deacylation of diacylglycerol. Lysosomal diacylglycerol hydrolysis is sensitive towards non-ionic detergents, cationic amphiphilic drugs and the lipase inhibitor RHC 80267.

Kinetic behavior of porcine pancreatic phospholipase A2 on zwitterionic and negatively charged double-chain substrates.

A number of isomeric diacylglycerophosphocholines and diacylglycero sulfates containing O-acyl and/or S-acyl ester bonds were investigated as substrates for porcine pancreatic phospholipase A2 and its zymogen. A comparison is made with the kinetic properties of the enzyme toward the corresponding glycol detergents previously described [van Oort, M. G., Dijkman, R., Hille, J. D. R., & de Haas, G. H. (1985) Biochemistry (preceding paper in this issue)]. Hydrolysis of the secondary ester bond in the 1,2-diacylglycero-3-type lipids proceeds much faster than the splitting of the primary ester function present in the isomeric 1,3-diacylglycerol and 1-acylglycol derivatives. In sharp contrast to the glycol detergents, the substitution of the cleavable oxygen ester by a thio ester bond in the glycerol lipids results in 5 times lower kcat values. At alkaline pH and above the critical micelle concentration, the anionic sulfates are much better substrates than the corresponding phosphocholine-containing detergents. At very low detergent concentrations, below the critical micelle concentration, the anionic sulfates induce protein aggregation such that phospholipase A2, as well as its zymogen, is present in high molecular weight complexes containing several protein molecules. In these aggregates, protein-protein and/or lipid-protein interactions strongly activate phospholipase but not the zymogen.

Studies on the substrate specificity of purified human milk lipoprotein lipase.

The fatty acid specificity of purified human milk lipoprotein lipase was studied using the C18 to C54 (total acyl carbon number) saturated and the C54 mono-, di- and triunsaturated monoacid triacylglycerols. Kinetic determinations indicated that the medium-chain triacylglycerols were better substrates than long- or very short-chain saturated triacylglycerols. The unsaturated triacylglycerols were hydrolyzed at rates comparable to that of tricaprylin with triolein having the highest rate of hydrolysis of the unsaturated species tested. The enzyme attacked the primary ester bond much more readily than the secondary ester bond. The purified human milk lipoprotein lipase showed a preferential stereospecific lipolysis of the sn-1-position of the triacylglycerol molecule.

Studies on the characterization of the sodium-potassium transport adenosine triphosphatase: IX. On the role of phospholipids in the enzyme

Incubation of a partially purified beef brain NaK ATPase preparation which is estimated to be 25–50% pure with protease-free phospholipase A led to a considerable loss in turbidity of the preparation and about a 90% inactivation of the NaK ATPase. Inactivation was retarded by Na and ATP. The secondary ester bonds were cleaved almost quantitatively. Evidence is presented that the inactivation of the NaK ATPase by phospholipase A was not due to the lysophosphatides which were liberated. If phospholipase A treatment was carried out in the presence of defatted serum albumin, almost complete reconstitution of enzyme activity could be achieved with a commercial preparation of soybean phosphatides—Inosithin. The specific activity of the reconstituted enzyme was one to two orders of magnitude higher than that reported in most of the earlier studies. Sixty to 80% reconstitution could be achieved with any one of the following phosphatides: phosphatidylserine, phosphatidylinositol, phosphatidic acid, or didodecylphosphate. Mixtures of these various phosphatides were no more effective than any one phosphatide alone. Conditions for reconstitution were examined in some detail. Reconstitution was more effective at 0 than at 37 °C, appeared to be almost instantaneous, was not affected by Na, K, Mg, or ATP and was rather insensitive to pH (optimum pH ranged from 6.0 to 7.5). After phospholipase A treatment, the reconstitutable protein was found to reside in the sedimentable fraction of the NaK ATPase preparation even though one-third to one-half of the protein had been solubilized by the phospholipase A treatment. At certain stages of purification of the NaK ATPase the phosphatidylserine content of the enzyme was enriched, the sphingomyelin disappeared and the phosphatidylcholine and phosphatidylethanolamine contents remained the same. Within the limits of sensitivity of the methods no phosphatidylinositol or phosphatidic acid could be detected in the most purified enzyme preparation. The evidence suggests that phosphatidylserine may be the phosphatide whose removal is responsible for the loss of NaK ATPase activity on incubation with phospholipase A but that other acidic phosphatides can substitute for phosphatidylserine.