Unusually versatile substrate specificity is shown by lipases. Not only do they hydrolyze triacylglycerols—for example, in the stomach and intestine during digestion of dietary fat—and various ...synthetic esters and amides, but their high stability in organic solvents permits their use in transesterification reactions and ester synthesis as well. Reactions based on lipase catalysis usually proceed with high regio‐ and enantioselectivity. Thus, the Ca2+ antagonist diltiazem (1) was obtained with lipase from Serratia marcescens. Over 30 lipases have been cloned in the last few years. Since the tertiary structure of 12 lipases is known, there are presently significant efforts to improve this class of enzymes by protein engineering techniques, in view of their use in detergents and other fields of industrial application.
Hormone-sensitive lipase (HSL) contributes importantly to the hydrolysis of cholesteryl ester in steroidogenic tissues, releasing the cholesterol required for adrenal steroidogenesis. HSL has broad ...substrate specificity, because it hydrolyzes triacylglycerols (TAGs), diacylglycerols, monoacylglycerols, and cholesteryl esters. In this study, we developed a specific cholesterol esterase assay using cholesterol oleate (CO) dispersed in phosphatidylcholine and gum arabic by sonication. To continuously monitor the hydrolysis of CO by HSL, we used the pH-stat technique. For the sake of comparison, the hydrolysis of CO dispersion was also tested using other cholesteryl ester-hydrolyzing enzymes. The specific activities measured on CO were found to be 18, 100, 27, and 3 μmol/min/mg for HSL, cholesterol esterase from Pseudomonas species, Candida rugosa lipase-3, and cholesterol esterase from bovine pancreas, respectively. The activity of HSL on CO is ∼4- to 5-fold higher than on long-chain TAGs. In contrast, with all other enzymes tested, the rates of TAG hydrolysis were higher than those of CO hydrolysis. The relatively higher turnover of HSL on CO observed in vitro adds further molecular insight on the physiological importance of HSL in cholesteryl ester catabolism in vivo.
Thus, HSL could be considered more as a cholesteryl ester hydrolase than as a TAG lipase.
The pancreas expresses several members of the lipase gene family including pancreatic triglyceride lipase (PTL) and two homologous proteins, pancreatic lipase-related proteins 1 and 2 (PLRP1 and ...PLRP2). Despite their similar amino acid sequences, PTL, PLRP1, and PLRP2 differ in important kinetic properties. PLRP1 has no known activity. PTL and PLRP2 differ in substrate specificity, bile acid inhibition, colipase requirement, and interfacial activation. To begin understanding the structural explanations for these functional differences, we solved the crystal structure of rat (r)PLRP2 and further characterized its kinetic properties. The 1.8 Å structure of rPLRP2, like the tertiary structure of human PTL, has a globular N-terminal domain and a β-sandwich C-terminal domain. The lid domain occupied the closed position, suggesting that rPLRP2 should show interfacial activation. When we reexamined this issue with tripropionin as substrate, rPLRP2 exhibited interfacial activation. Because the active site topology of rPLRP2 resembled that of human PTL, we predicted and demonstrated that the lipase inhibitors E600 and tetrahydrolipstatin inhibit rPLRP2. Although PTL and rPLRP2 have similar active sites, rPLRP2 has a broader substrate specificity that we confirmed using a monolayer technique. With this assay, we showed for the first time that rPLRP2 prefers phosphatidylglycerol and ethanolamine over phosphatidylcholine. In summary, we confirmed and extended the observation that PLRP2 lipases have a broader substrate specificity than PTL, we demonstrated that PLRP2 lipases show interfacial activation, and we solved the first crystal structure of a PLRP2 lipase that contains a lid domain.
This article reviews our present knowledge on the extracellular lipolytic enzymes LipA and LipB from
Bacillus subtilis. Growth of
B. subtilis to the late logarithmic growth phase results in a total ...lipolytic activity of 12–18 units per liter of culture supernatant. Immunodetection with LipA- and LipB-specific antibodies indicated a differential expression of both lipolytic enzymes depending on the composition of the growth medium. LipA was produced in rich and in minimal medium, whereas LipB was present only in rich medium. The
lipA and
lipB genes were cloned and overexpressed in
B. subtilis and
Escherichia coli, the corresponding proteins purified to electrophoretic homogeneity and their substrate specificities, pH- and temperature stabilities were determined. The active site residue Ser
78 of LipB is located in the consensus sequence Ala–X–Ser–X–Gly where the alanine replaces a glycine found in most of the bacterial lipases. The role of this Ala-residue was investigated by constructing LipB variant A76G thereby restoring the canonical lipase consensus motif. When compared with wild-type LipB this variant showed a markedly reduced thermostability at pH 11 but an increased stability at pH 5–7. These findings were rationalized by building a three-dimensional structural model of LipB using the atomic coordinates of the LipA crystal structure, which was solved recently. The LipB model structure revealed that 43 out of 45 residues, which are different from LipA, were located on the surface of LipB. The surface-exposed amino acids including those located at the rim of the active site cleft may cause the differences in specific activities between LipA and LipB.
Five key amino acid residues from human pancreatic lipase (HPL) are mutated in some pancreatic lipase-related proteins 2 (PLRP2) that are not reactivated by colipase in the presence of bile salts. ...One of these residues (Y403) is involved in a direct interaction between the HPL C-terminal domain and colipase. The other four residues (R256, D257, Y267, and K268) are involved in the interactions stabilizing the open conformation of the lid domain, which also interacts with colipase. Here we produced and characterized three HPL mutants: HPL Y403N, an HPL four-site mutant (R256G, D257G, Y267F, and K268E), and an HPL five-site mutant (R256G, D257G, Y267F, K268E, and Y403N), in which the HPL amino acids were replaced by those present in human PLRP2. Colipase reactivated both the HPL Y403N mutant and HPL, and Y403 is therefore not essential for lipase−colipase interactions. Both the HPL four-site and five-site mutants showed low activity on trioctanoin, were inhibited by bile salts (sodium taurodeoxycholate, NaTDC) and were not reactivated by colipase. The interfacial binding of the HPL four-site mutant to a trioctanoin emulsion was suppressed in the presence of 4 mM NaTDC and was not restored by addition of colipase. Protein blotting/protein overlay immunoassay revealed that the HPL four-site mutant−colipase interactions are not abolished, and therefore, the absence of reactivation of the HPL four-site mutant is probably due to a lid domain conformation that prevents the interfacial binding of the lipase−colipase complex. The effects of colipase were also studied with HPL(−lid), an HPL mutant showing an 18-residue deletion within the lid domain, which therefore has only one colipase interaction site. HPL(−lid) showed a low activity on trioctanoin, was inhibited by bile salts, and recovered its lipase activity in the presence of colipase. Reactivation of HPL(−lid) by colipase was associated with a strong interfacial binding of the mutant to a trioctanoin emulsion. The lid domain is therefore not essential for either the interfacial binding of HPL or the lipase−colipase interactions.
The enzymes secreted in the intercellular spaces of stratum corneum (SC), the outermost layer of the epidermis, are thought to be involved in normal desquamation and skin barrier function. Their ...activity can barely be measured due to the difficulty in isolating enough biological material. Human SC layers were obtained from the forearm of healthy volunteers by the tape stripping technique. Assays for esterase activities were carried out in specially designed plates which contained the SC blotted on tape strips, using various fluorescent methylumbelliferone acyl esters as substrates. Triacylglycerol hydrolase activities were also studied by this method. By using radiolabeled triolein and fluorescent 4-methylumbelliferyl 7-oleate as substrates, true lipase activities could be detected and quantitated in SC at pH 5.5 and 7.5. These activities were shown to be strongly inhibited by tetrahydrolipstatin while this was not the case with 4-methylumbelliferyl 7-heptanoate. The method described here combines the painless tape stripping technique with a sensitive plate assay analysis. Since the whole process needs little manipulation, this method can permit rapid quantitation of multiple enzyme activities from a single strip. Therefore, it will permit the study of the involvement of enzyme activities in epidermis aging and skin pathologies.
In the present paper, a study on the stereoselectivity of 25 lipases of animal and microbial origin towards homogeneous prochiral triglycerides is presented. All the lipases tested catalyse the ...hydrolysis of the chemically alike but sterically nonequivalent ester groups in trioctanoin and triolein with different degrees of stereobias, depending on the fatty acyl chain length of the substrate (Rogalska et al., J. Biol. Chem. 256:20271-20276, 1990). Hydrolysis of the sn-2 ester group is catalysed by very few lipases and only Candida antarctica A shows a clear preference for this position. Most of the lipases investigated (12 with trioctanoin and 16 with triolein) showed a preference for the sn-1 position. Using trioctanoin as substrate we observed a total stereoselectivity for position sn-1 with Pseudomonas sp. and Pseudomonas aeruginosa and for position sn-3 with Candida antarctica B. This was not the case with triolein as substrate. Among the 23 lipases studied here and the other two lipases described previously (Rogalska et al., J. Biol. Chem. 256:20271-20276, 1990), 17 show a higher stereoselectivity with trioctanoin than with triolein. With guinea pig pancreatic lipase and with three mold lipases (Geotrichum candidum M, Geotrichum candidum A, and Candida antarctica B), the preference switches from sn-3 to sn-1 when the acyl chain length increases from eight to 18 carbon atoms. The main conclusion to emerge from the present study is that the specific stereopreference of each lipase for a given substrate under given lipolytic conditions can be said to be its fingerprint.
The ability of Chicken PLA2 group IIA to hydrolyse pure 1,2 DDPC contrary to the human one. Display omitted
► The interfacial kinetic and binding data for the pancreatic and intestinal sPLA2 from ...bird and mammals. ► PC, in contrast to PE and PG, were resistant to the hydrolysis by hPLA2-IIA. ► ChPLA2-IIA was found to be able to hydrolyse all phospholipids. ► The binding of sPLA2 at the lipid–water interface is governed by the electrostatic and hydrophobic forces.
The interfacial kinetic and binding data for the pancreatic and intestinal sPLA2 from bird and mammals show that these enzymes have dramatically different ability to bind and hydrolyse phospholipids. The main conclusions from our experimental data indicate that phosphatidylcholine monolayers (PC), in contrast to phosphatidylethanolamine (PE) and phosphatidylglycerol (PG), were resistant to the hydrolysis by human intestinal sPLA2. Conversely, chicken intestinal sPLA2 was found to be able to hydrolyse all the phospholipids tested, including PC. The experiments show also that the interfacial penetrating ability of chicken sPLA2 (from intestine and pancreas) was higher than their mammalian’s orthologs. This observation is confirmed by the activity of pancreatic chicken PLA2 measured on PC film showing that the interfacial pressure window that permits sPLA2 activity was very large, between 5 and 20dynescm−1, compared with the porcine pancreatic sPLA2-IB which was inactive at pressure above 15dynescm−1.
In trying to establish a structure–function relationship, we examined the surface electrostatic potentials of the various sPLA2 from chicken and mammals. We reported in this study that the binding, orientation and persistence of sPLA2 at the lipid–water interface is probably governed by the electrostatic and hydrophobic forces operative at this surface. These variations argue strongly that these enzymes are not isoforms and that they are expected to have functions other than the release of lipid mediators for the biosynthesis of the eicosanoids.
We have designed a convenient, specific, sensitive and continuous lipase assay based on the use of natural triacylglycerols (TAGs) from the
Aleurites fordii
seed oil which contains α-eleostearic acid ...(9,11,13,
cis
,
trans
,
trans
-octadecatrienoic acid) and which was coated in the wells of microtiter plates. The coated TAG film cannot be desorbed by the various buffers used during the lipase assay. Upon lipase action, α-eleostearic acid is liberated and desorbed from the interface and then solubilized into the micellar phase. Consequently, the UV absorbance of the α-eleostearic acid is considerably enhanced due to the transformation from an adsorbed to a water soluble state. The lipase activity can be measured continuously by recording the variations with time of the UV absorption spectra. The rate of lipolysis was monitored by measuring the increase of OD at 272 nm, which was found to be linear with time and directly proportional to the amount of added lipase. This microtiter plate lipase assay, based on coated TAGs, presents various advantages as compared to the classical systems: (i) coated TAGs on the microtiter plates could be stored for a long-time at 4 °C, (ii) higher sensitivity in lipase detection, (iii) good reproducibility, and (iv) increase of signal to noise ratio due to high UV absorption after transfer of α-eleostearic acid from an adsorbed to a soluble state. Low concentrations, down to 1 pg mL
−1
of pure
Thermomyces lanuginosus
or human pancreatic lipase, could be detected under standard assay conditions. The detection sensitivity of this coated method is around 1000 times higher as compared to those obtained with the classical emulsified systems. This continuous high throughput lipase assay could be used to screen new lipases and/or lipase inhibitors present in various biological samples.
We have designed a convenient, specific, sensitive and continuous lipase assay based on the use of natural triacylglycerols (TAGs) from the
Aleurites fordii
seed oil which contains α-eleostearic acid and which was coated in the wells of microtiter plates.
Rhizopus oryzae lipase (ROL) was found to be a true lipase. This enzyme presents the interfacial activation phenomenon. The N-terminal amino acid sequence of ROL was compared to those of rhizopus ...lipases. Purified ROL possesses the same N-terminal sequence as the mature
Rhizopus niveus lipase (RNL). This sequence was found in the last 28 amino acids of the propeptide sequence derived from the cDNA of
Rhizopus delemar lipase (RDL). Using the baro-stat method, we have measured the hydrolysis rate of dicaprin films by ROL as a function of surface pressure. Our results show that
Rhizopus oryzae lipase is markedly stereoselective of the
sn-3 position of the 2,3 enantiomer of dicaprin. Polyclonal antibodies (PAB) directed against ROL have been produced and purified by immunoaffinity. The effects of these PAB on the interfacial behavior of ROL were determined. The immunoblot analysis with polyclonal antibodies anti-ROL (PAB anti-ROL) and various lipases shows a cross-immunoreactivity between the lipase from the rhizopus family (
Rhizopus delemar lipase and
Rhizopus arrhizus lipase).
PDF
