Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “LIPASE”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 937 records · Page 52Linked to original sources
PubMed1996

Enantiomeric perylene-glycerolipids as fluorogenic substrates for a dual wavelength assay of lipase activity and stereoselectivity.

A new type of fluorogenic alkyldiacyl glycerols was synthesized and used as fluorogenic substrates for the analysis of lipase activities and stereoselectivities. These compounds contain perylene as a fluorophore and the trinitrophenylamino (TNP) residue as a quencher. Both substituents are covalently bound to the omega-ends of the sn-2 and sn-1 (3) acyl chains, respectively. Upon glycerolipid hydrolysis, the residues are separated from each other thus allowing determination of lipase activity by the continuous increase in fluorescence intensity which is caused by dequenching. Using enantiomeric pairs of these compounds, we were able to analyze lipase stereoselectivity depending on the reaction medium. Mixtures of enantiomeric fluorogenic alkyldiacyl glycerols, selectively labelled with pyrene or perylene as fluorophores, can be used for a dual-wavelength "stereoassay" of lipases. Since absorption and emission maxima of both labels are clearly separated, hydrolysis of the respective enantiomeric substrates can be determined simultaneously, and the difference in the rates of hydrolysis can be taken as a parameter for the stereopreference of a lipase. Hydrolysis rates measured with perylene-substituted lipids are generally lower than those obtained with the pyrene analogs. Thus, with a mixture of perylene and pyrene-substituted lipids, we observe a higher apparent stereoselectivity of lipases since we measure a combination of stereo- and substrate selectivity. In the presence of albumin, all microbial lipases tested so far exhibit stereopreference for the sn-1 glycerol position. In our assay, the apparent stereoselectivities are highest if in the presence of albumin, the sn-1 position carries pyrene and the sn-3 position is substituted with perylene. The lipase stereoselectivity assay described here requires the simultaneous measurement of the fluorescence intensities at two different wavelengths in a single cuvette and can thus be carried out using existing and cheap instrumentation that was developed for the fluorimetric analysis of Ca+2 concentrations.

Animals↗
PubMed1990

Lipoprotein and hepatic lipase activity and high-density lipoprotein subclasses after cardiac transplantation.

Atherosclerosis is the leading obstacle to long-term survival in cardiac transplant patients. Increases in plasma triglycerides and lipoprotein cholesterol levels occur after transplantation that may contribute to transplant atherosclerosis. The etiology of this increase is unclear. We investigated the interaction of immunosuppressive medications with plasma triglycerides, low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol, the HDL subclasses HDL2 and HDL3 cholesterol, and hepatic and lipoprotein lipase activity in 72 consecutive cardiac transplant patients compared to 51 healthy control subjects. In the transplantation group, greater concentrations of plasma triglyceride (80%, p less than 0.001), LDL cholesterol (16%, p less than 0.005) and hepatic lipase activity (100%, p less than 0.001) were noted, whereas lipoprotein lipase activity was noted to be significantly lower (124%, p less than 0.001). No difference was detected in HDL, HDL2, or HDL3 cholesterol. Cyclosporine dose was significantly associated with hepatic lipase activity (r = 0.33, p less than 0.02) and inversely associated with lipoprotein lipase activity (r = -0.28, p less than 0.05). Lipoprotein lipase activity after transplantation correlated inversely with triglycerides (r = -0.36, p less than 0.002) and positively with HDL cholesterol (r = 0.23, p less than 0.05) and HDL2 cholesterol (r = 0.29, p less than 0.05). Hepatic lipase activity correlated inversely with LDL cholesterol (r = -0.21, p less than 0.08). In multiple regression analysis, cyclosporine dose was the major source of variation in hepatic lipase activity.

Adult↗
PubMedDate not supplied

A comparative study of human tissue and post-heparin plasma triglyceride lipases.

Human post-heparin plasma contains at least two different triglyceride lipases (TGL). The plasma lipolytic activity has been attributed to extra-hepatic and hepatic origin. Both post-heparin triglyceride lipases were partially purified and characterized. With heparin-Sepharose 4 B affinity chromatography it was possible to partially purify human adipose tissue lipoprotein lipase (LPL) as well as a lipase from human liver. The effects of NaCl, pre-heparin plasma, pH and temperature on these two tissue lipases and plasma lipases were studied in parallel. Antibodies were produced against plasma hepatic triglyceride lipase (plasma H-TGL) that did not cross react with LPL. TGL activity of human liver was completely inhibited by antibodies against plasma H-TGL. From these results it appears that human post-heparin plasma contains two triglyceride lipase activities which originate from liver and extra-hepatic tissues such as adipose tissue.

Adipose Tissue↗
PubMed1994

A structural model of mono- and diacylglycerol lipase from Penicillium camembertii.

The amino acid sequence of lipase from Penicillium camembertii was aligned with Rhizomucor miehei lipase without permitting any deletion or insertion in the structurally conserved regions. This lipase was classified into the R. miehei lipase family, because 33% of the residues were identical and 18% of the exchanges were conserved. A graphic molecular model for P. camembertii lipase was built using information from the sequence and X-ray structure of R. miehei lipase. The primary specificity pocket in the model of P. camembertii lipase predicted a substrate preference for monoacylglycerols and diacylglycerols. The close region to reactive His259 in P. camembertii lipase, which located in the opposite shore to the helical lid that was predictable to move in the activated state, contributed to the decision of the unique substrate specificity.

Amino Acid Sequence↗
PubMed2003

Inhibitory effects of grape seed extract on lipases.

OBJECTIVE: The aim of the present study was to assess the effects of grape seed extract (GSE) on the fat-metabolizing enzymes pancreatic lipase, lipoprotein lipase, and hormone-sensitive lipase in vitro and evaluate its potential application as a treatment for obesity. METHODS: Crushed grape seeds were extracted in ethanol, and the extract was assayed for the measurement of inhibitory effects on pancreatic lipase and lipoprotein lipase activities and on lipolysis of 3T3-L1 adipocytes. RESULTS: The GSE rich in bioactive phytochemicals showed inhibitory activity on the fat-metabolizing enzymes pancreatic lipase and lipoprotein lipase, thus suggesting that GSE might be useful as a treatment to limit dietary fat absorption and the accumulation of fat in adipose tissue. The observed reduction in intracellular lipolytic activity of cultured 3T3-L1 adipocytes may reduce the levels of circulating free fatty acids that have been linked to insulin resistance in obese patients. CONCLUSION: The GSE rich in compounds that inhibit lipases may provide a safe, natural, and cost-effective weight control treatment.

Adipose Tissue↗
PubMed1992

Functional topology of a surface loop shielding the catalytic center in lipoprotein lipase.

Lipoprotein lipase (LPL), hepatic lipase, and pancreatic lipase show high sequence homology to one another. The crystal structure of pancreatic lipase suggests that it contains a trypsin-like Asp-His-Ser catalytic triad at the active center, which is shielded by a disulfide bridge-bounded surface loop that must be repositioned before the substrate can gain access to the catalytic residues. By sequence alignment, the homologous catalytic triad in LPL corresponds to Asp156-His241-Ser132, absolutely conserved residues, and the homologous surface loop to residues 217-238, a poorly conserved region. To verify these assignments, we expressed in vitro wild-type LPL and mutant LPLs having single amino acid mutations involving residue Asp156 (to His, Ser, Asn, Ala, Glu, or Gly), His241 (to Asn, Ala, Arg, Gln, or Trp), or Ser132 (to Gly, Ala, Thu, or Asp) individually. All 15 mutant LPLs were totally devoid of enzyme activity, while wild-type LPL and other mutant LPLs containing substitutions in other positions were fully active. We further replaced the 22-residue LPL loop which shields the catalytic center either partially (replacing 6 of 22 residues) or completely with the corresponding hepatic lipase loop. The partial loop-replacement chimeric LPL was found to be fully active, and the complete loop-replacement mutant had approximately 60% activity, although the primary sequence of the hepatic lipase loop is quite different. In contrast, replacement with the pancreatic lipase loop completely inactivated the enzyme. Our results are consistent with Asp156-His241-Ser132 being the catalytic triad in lipoprotein lipase.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed2002

Effect of oral oleoyl-estrone treatment on plasma lipoproteins and tissue lipase activities of Zucker lean and obese female rats.

OBJECTIVE: To study the effect of oral oleoyl-estrone on the plasma lipoprotein profile and tissue lipase activities in order to determine the handling of circulating lipids by adipose tissue, liver and muscle of obese female rats. DESIGN: Lean (Fa/?) and obese (fa/fa) female Zucker rats treated for 10 days with a daily gavage of 0.2 ml sunflower oil containing 0 (controls) or 10 micromol/kg of oleoyl-estrone. After sacrifice, samples of tissues and plasma were taken. MEASUREMENTS: Plasma lipoprotein classes and composition; lipoprotein lipase and hepatic lipase activities in plasma, liver, skeletal muscle and periovaric and mesenteric white adipose tissue (WAT). RESULTS: Oleoyl-estrone decreased plasma cholesterol (mainly in HDLs: 76%) of lean rats, but dramatically decreased all lipid classes in obese rats, in which chylomicra and VLDL lost most of their triacylglycerols (95 and 81%, respectively). Hepatic lipase activity decreased markedly with oleoyl-estrone in all groups, both in plasma (79% lean, 100% obese) and liver (62% in both groups). Lipoprotein lipase activity was largely unchanged by oleoyl-estrone in lean rats, but in the obese it decreased in WAT (82% in periovaric, and 49% in mesenteric), and increased in plasma (x4) and in skeletal muscle (x5); liver levels showed no change. CONCLUSIONS: The shift observed in obese rats from a decrease in liver and WAT lipoprotein lipase and hepatic lipase activities to an increase in muscle lipoprotein lipase is coincident with the hypolipemic effect of oleoyl-estrone, especially in obese rats, and indicates that muscle is a key site for the disposal of endogenous fat mobilized due to oleoyl-estrone treatment.

Adipose Tissue↗
PubMed1980

Interaction of lipoprotein lipase with native and modified heparin-like polysaccharides.

1. Lipoprotein lipase (EC 3.1.1.34), which was previously shown to bind to immobilized heparin, was now found to bind also to heparan sulphate and dermatan sulphate and to some extent to chondroitin sulphate. 2. The relative binding affinities were compared by determining (a) the concentration of NaCl required to release the enzyme from polysaccharide-substituted Sepharose; (b) the concentration of free polysaccharides required to displace the enzyme from immobilized polysaccharides; and (c) the total amounts of enzyme bound after saturation of immobilized polysaccharides. By each of these criteria heparin bound the enzyme most efficiently, followed by heparan sulphate and dermatan sulphate, which were more efficient than chondroitin sulphate. 3. Heparin fractions with high and low affinity for antithrombin, respectively, did not differ with regard to affinity for lipoprotein lipase. 4. Partially N-desulphated heparin (40-50% of N-unsubstituted glucosamine residues) was unable to displace lipoprotein lipase from immobilized heparin. This ability was restored by re-N-sulphation or by N-acetylation; the N-acetylated product was essentially devoid of anticoagulant activity. 5. Partial depolymerization of heparin led to a decrease in ability to displace lipoprotein lipase from heparin-Sepharose; however, even fragments of less than decasaccharide size showed definite enzyme-releasing activity. 6. Studies with hepatic lipase (purified from rat post-heparin plasma) gave results similar to those obtained with milk lipoprotein lipase. However, the interaction between the hepatic lipase and the glycosaminoglycans was weaker and was abolished at lower concentrations of NaCl. 7. The ability of the polysaccharides to release lipoprotein lipase to the circulating blood after intravenous injection into rats essentially conformed to their affinity for the enzyme as evaluated by the experiments in vitro.

Animals↗
PubMed1963

PRODUCTION OF LIPASE BY PSEUDOMONAS FRAGI IN A SYNTHETIC MEDIUM.

Alford, John A. (U.S. Department of Agriculture, Beltsville, Md.) and David A. Pierce. Production of lipase by Pseudomonas fragi in a synthetic medium. J. Bacteriol. 86:24-29. 1963.-The growth and production of lipase by Pseudomonas fragi in 41 dehydrated media were determined. From a chemical analysis of the peptone medium which gave the maximal lipase production, numerous synthetic media were prepared. Good cell growth, but no lipase production, was obtained on a buffered ammonium sulfate-glucose medium. The addition of arginine, lysine, aspartic acid, and glutamic acid to this medium gave lipase production approaching that obtained in the peptone medium. Omission of the glucose had little effect on growth, but no lipase were produced. Sterilization of the glucose with either the amino acids or phosphate buffer had no effect on growth, but sharply reduced lipase production. Gas chromatographic analysis of the fatty acids liberated from lard, corn oil, and coconut oil indicated that medium composition had no effect on enzyme specificity. P. fluorescens produced very little lipase in any of the synthetic media examined.

Adenine↗
PubMed1984

Lipid profiles and lipase activities in children and adolescents with chronic renal failure treated conservatively or with hemodialysis or transplantation.

To elucidate the pathogenesis of hyperlipidemia in chronic renal disease in children and adolescents, we have measured serum triglyceride, total cholesterol, high density lipoprotein cholesterol (HDL-C) and activities of postheparin plasma lipoprotein lipase and hepatic triglyceride lipase (EC 3.1.1.3) in nine patients with transplants, and nine hemodialyzed and 18 conservatively treated patients with chronic renal failure. In 29 of 36 patients, serum insulin levels both in fasting and in response to oral glucose load were measured. The lipase activities were measured separately, utilizing antiserum against hepatic triglyceride lipase. All groups of patients had hypertriglyceridemia. The patients with endogenous creatinine clearance less than 20 ml/min/m2 had a low HDL-C level. The HDL-C level was correlated inversely with serum triglyceride level and positively with glomerular filtration rate. The lipoprotein lipase activities were low in patients with endogenous creatinine clearance less than 20 ml/min/m2. Although hepatic triglyceride lipase activities were not significantly low in any groups of patients, they were correlated with glomerular filtration rates in the conservatively treated patients with chronic renal failure. A defective triglyceride removal due to low lipase activities may contribute to uremic hypertriglyceridemia in these patients. On the other hand, patients with transplants had almost normal lipase activities and exhibited hyperinsulinemia; overproduction of triglyceride due to hyperinsulinemia may contribute to their hypertriglyceridemia.

Adolescent↗
PubMed2002

Diurnal variation in lipoprotein lipase activity.

BACKGROUND: We investigated whether variations in lipoprotein lipase activity, a key post-prandial enzyme involved in the removal of circulating dietary triglycerides, could contribute to the previously described nocturnal lipid intolerance. METHODS: We studied lipoprotein lipase activity in 12 healthy volunteers (five women, seven men) at 11:30 h and 23:30 h on two separate occasions. Subjects consumed a high-fat mixed meal at 07:30 h for the morning study or 19:30 h for the evening study. Then, after a 4-h fast, subjects were given an intravenous bolus of 7,500 U heparin. Blood samples were collected before and 15 min after heparin administration for measurement of lipoprotein lipase, hepatic lipase, triglycerides and non-esterified fatty acids concentrations. RESULTS: Post-prandial post-heparin lipoprotein lipase activity was greater in the morning than in the evening (16.5 +/- 1.4 versus 14.4 +/- 1.0 micromol oleate/mL/h; P< 0.05). Post-prandial post-heparin hepatic lipase activity was also greater in the morning than in the evening (8.7 +/- 1.5 versus 81 +/- 1.6 micromol oleate/mL/h; P= 0.002). There were no other significant diurnal differences. CONCLUSION: We report a diurnal variation in post-prandial lipoprotein lipase activity. This is consistent with the notion that decreased nocturnal insulin sensitivity extends to insulin's actions on lipoprotein lipase and provides a possible explanation for nocturnal lipid intolerance.

Adult↗
PubMed2004

Inhibition of lipase activity and lipolysis in rat islets reduces insulin secretion.

Lipids may serve as coupling factors in K(ATP)-independent glucose sensing in beta-cells. We have previously demonstrated that beta-cells harbor lipase activities, one of which is the hormone-sensitive lipase. Whether beta-cell lipases are critical for glucose-stimulated insulin secretion (GSIS) by providing lipid-derived signals from endogenous lipids is unknown. Therefore, using a lipase inhibitor (orlistat), we examined whether lipase inhibition reduces insulin secretion. Islet lipolysis stimulated by glucose and diglyceride lipase activity was abolished by orlistat. Incubation of rat islets with orlistat dose dependently inhibited GSIS; this inhibition was reversed by 1 mmol/l palmitate, suggesting that orlistat acts via impaired formation of an acylglyceride-derived coupling signal. Orlistat inhibited the potentiating effect of forskolin on GSIS, an effect proposed to be due to activation of a lipase. In perifused islets, orlistat attenuated mainly the second phase of insulin secretion. Because the rise in islet ATP/ADP levels in response to glucose and oxidation of the sugar were unaffected by orlistat whereas the second phase of insulin secretion was reduced, it seems likely that a lipid coupling factor involved in K(ATP)-independent glucose sensing has been perturbed. Thus, beta-cell lipase activity is involved in GSIS, emphasizing the important role of beta-cell lipid metabolism for insulin secretion.

Adipocytes↗
PubMed1976

Human milk lipases and their possible role in fat digestion.

Human milk contains two lipases. One is a lipoprotein lipase with properties similar to the lipoprotein lipases that participate in the metabolism of blood plasma lipoproteins in several tissues. This enzyme is present in high activity in the lactating mammary gland where it facilitates the uptake of triglyceride fatty acids from the blood lipoproteins for production of milk lipids in the gland. The high activity of this enzyme in milk probably represent leakage of enzyme from the gland. This lipase is not stable at pH below 5 or in intestinal contents and it is unlikely that it participates in intestinal fat digestion. Its activity varies widely between individual milk samples, and there is a high correlation between its activity and the development of hydrolytic rancidity in the milk on storage. The other lipase is present in the milk in an inactive form which is activated by bile salts. This lipase is present in milk from primates but not in milk from lower animals. Human milk contains enough of this lipase to hydrolyze the milk lipids almost completely in less than half an hour at the pH and the bile acid and salt concentrations found in the small intestine of the human infant. It is probable that it increases the efficiency of milk fat absorption. The enzyme has a rather wide substrate specificity and may also act on other lipid substrates than triglycerides. In contrast to pancreatic lipase it hydrolyses all three ester bonds in a triglyceride. This may affect the physical chemistry of the lipids in the intestinal contents as well as their absorption and further metabolism in the musoca.

Bile Acids and Salts↗
PubMed2000

Enzymatic resolution to (-)-ormeloxifene intermediates from their racemates using immobilized Candida rugosa lipase.

In the synthesis of (-)-ormeloxifene, a drug candidate recently under development, enzymatic resolution of potential intermediates can be carried out using a simple, practical method. Five commercially available lipases, Candida rugosa lipase, Candida antarctica lipase B, Mucor miehei lipase, Pseudomonas cepacia lipase, and Humicola lanuginosa lipase, all immobilized on Accurel(R), were initially screened in combination with four different substrates belonging to the class of phenyl esters. Excellent stereoselectivity was observed using C. rugosa lipase with an acetate as substrate, but low reaction rates were observed in scale-up experiments. However, by changing the acyl part of the ester into a hexanoyl moiety and subjecting this substrate to enzymatic hydrolysis in aqueous acetonitrile at room temperature by C. rugosa lipase, it became possible to run the reaction to a 50% conversion on a 10 g scale within a period of 4 h, obtaining a phenolic product of more than 95% ee that could be converted to the target molecule, (-)-ormeloxifene, in two synthetic steps. Simple recovery of the immobilized enzyme by filtration allowed multiple recycling of the catalyst without significant loss of enzymatic activity. Capillary electrophoresis with sulfobutyl ether beta-cyclodextrin as a chiral buffer additive and acetonitrile as an organic modifier was demonstrated to provide an excellent chiral analytical tool for monitoring the enzymatic reactions.

Antifungal Agents↗
PubMed2001

Production of Acinetobacter radioresistens lipase with repeated batch culture in presence of nonwoven fabric.

Cultivation of Acinetobacter radioresistens on n-hexadecane for lipase production was investigated with repeated batch culture in the presence of a hydrophobic nonwoven fabric. Lipase production followed the growth-associated model, and the repeated batch culture could achieve both high enzyme yield and increased volumetric productivity. The fabric was shown to be able to disperse n-hexadecane, to adsorb the unused hydrocarbon, and to retain bioemulsifiers excreted from the cells; therefore, it enhanced cell growth and, in turn, lipase production. In the repeated batch culture in the absence of the fabric, lipase yield and volumetric productivity were found to be 21 U/mL and 875 U/L. h, respectively. However, if the fabric was equipped in the fermentor, lipase yield and volumetric productivity increased to 30 U/mL and 2500 U/L. h, respectively. The lipase production profile could be further improved by raising the amount of nitrogen source and, as a result, a lipase yield of 54 U/mL and a volumetric productivity of 2250 U/L. h were obtained. In this study we assess the beneficial effects of nonwoven fabric on lipase production.

Acinetobacter↗
PubMed2004

Hyperactivation of Rhizomucor miehei lipase by hydrophobic xerogels.

Although a variety of approaches exist for the immobilization of enzymes, the "science" of enzyme immobilization is still in its infancy. In recent years, considerable interest has developed regarding the use of xerogels for enzyme immobilization. There are several advantages to xerogels for enzyme immobilization, including the opportunity to produce them in defined shapes or thin films and the ability to manipulate their physical characteristics (e.g., porosity, hydrophobicity, and optical properties). In this study we examined the effect of xerogel hydrophobicity on the activity of lipase (EC 3.2.2.3) from Rhizomucor miehei. The hydrophobicity of the xerogels was manipulated by generating xerogels with various molar ratios of propyltrimethoxysilane (PTMS) to tetramethoxysilane (TMOS), from 1:1 to 10:1. The belief was that, by increasing the proportion of propyl groups, the hydrophobicity of the resulting xerogel would be increased. Differences in the hydrophobicity of the resulting xerogels were confirmed using water-affinity studies. Two approaches were taken for water-affinity determinations by examining the ability of the xerogels to remove water from air (controlled humidity) and from water-saturated isopropyl ether. Xerogels with higher propyl content showed a reduced affinity for water. A crude lipase preparation from Rhizomucor miehei was then contacted with sized xerogel particulates and the effect of the xerogel on lipase activity was determined. The presence of the xerogel resulted in hyperactivation of the lipase. Analysis of the protein adsorption revealed changes in the profile of proteins adsorbed to the xerogel based on the hydrophobicity of the xerogel. Based on estimations of the specific activity of the hyperactivated lipase, a minimum hyperactivation of 207% was observed. Part of the hyperactivation may be attributable to xerogel-lipase interactions, but also to the adsorption of a component from the crude lipase preparation that may complex with the lipase and the xerogel producing a stabilizing effect. Further improvements in hyperactivation and selectivity of the xerogels is likely possible by working at lower PTMS:TMOS ratios than those investigated in this study.

Adsorption↗
PubMed2005

Display of lipase on the cell surface of Escherichia coli using OprF as an anchor and its application to enantioselective resolution in organic solvent.

We have developed a new cell surface display system using a major outer membrane protein of Pseudomonas aeruginosa OprF as an anchoring motif. Pseudomonas fluorescens SIK W1 lipase gene was fused to the truncated oprF gene by C-terminal deletion fusion strategy. The truncated OprF-lipase fusion protein was successfully displayed on the surface of Escherichia coli. Localization of the truncated OprF-lipase fusion protein was confirmed by western blot analysis, immunofluorescence microscopy, and whole-cell lipase activity. To examine the enzymatic characteristics of the cell surface displayed lipase, the whole-cell enzyme activity and stability were determined under various conditions. Cell surface displayed lipase showed the highest activity at 37 degrees C and pH 8.0. It retained over 80% of initial activity after incubation for a week in both aqueous solution and organic solvent. When the E. coli cells displaying lipases were used for enantioselective resolution of racemic 1-phenylethanol in hexane, (R)-phenyl ethyl acetate was successfully obtained with the enantiomeric excess of greater than 96% in 36 h of reaction. These results suggest that E. coli cells displaying lipases using OprF as an anchoring motif can be employed for various biotechnological applications both in aqueous and nonaqueous phases.

Bacterial Outer Membrane Proteins↗
PubMed2004

Characterization of covalently inhibited extracellular lipase from Streptomyces rimosus by matrix-assisted laser desorption/ionization time-of-flight and matrix-assisted laser desorption/ionization quadrupole ion trap reflectron time-of-flight mass spectrometry: localization of the active site serine.

A chemical modification approach combined with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry was used to identify the active site serine residue of an extracellular lipase from Streptomyces rimosus R6-554W. The lipase, purified from a high-level overexpressing strain, was covalently modified by incubation with 3,4-dichloroisocoumarin, a general mechanism-based serine protease inhibitor. MALDI time-of-flight (TOF) mass spectrometry was used to probe the nature of the intact inhibitor-modified lipase and to clarify the mechanism of lipase inhibition by 3,4-dichloroisocoumarin. The stoichiometry of the inhibition reaction revealed that specifically one molecule of inhibitor was bound to the lipase. The MALDI matrix 2,6-dihydroxyacetophenone facilitated the formation of highly abundant [M + 2H](2+) ions with good resolution compared to other matrices in a linear TOF instrument. This allowed the detection of two different inhibitor-modified lipase species. Exact localization of the modified amino acid residue was accomplished by tryptic digestion followed by low-energy collision-induced dissociation peptide sequencing of the detected 2-(carboxychloromethyl)benzoylated peptide by means of a MALDI quadrupole ion trap reflectron TOF instrument. The high sequence coverage obtained by this approach allowed the confirmation of the site specificity of the inhibition reaction and the unambiguous identification of the serine at position 10 as the nucleophilic amino acid residue in the active site of the enzyme. This result is in agreement with the previously obtained data from multiple sequence alignment of S. rimosus lipase with different esterases, which indicated that this enzyme exhibits a characteristic Gly-Asp-Ser-(Leu) motif located close to the N-terminus and is harboring the catalytically active serine residue. Therefore, this study experimentally proves the classification of the S. rimosus lipase as GDS(L) lipolytic enzyme.

Amino Acid Sequence↗