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Results for “Thiolester Hydrolases”

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At least 19 recordsLinked to original sources
PubMed2005

A promiscuous glutathione transferase transformed into a selective thiolester hydrolase.

Human glutathione transferase A1-1 (hGST A1-1) can be reengineered by rational design into a catalyst for thiolester hydrolysis with a catalytic proficiency of 1.4 x 10(7) M(-1). The thiolester hydrolase, A216H that was obtained by the introduction of a single histidine residue at position 216 catalyzed the hydrolysis of a substrate termed GSB, a thiolester of glutathione and benzoic acid. Here we investigate the substrate requirements of this designed enzyme by screening a thiolester library. We found that only two thiolesters out of 18 were substrates for A216H. The A216H-catalyzed hydrolysis of GS-2 (thiolester of glutathione and naphthalenecarboxylic acid) exhibits a k(cat) of 0.0032 min(-1) and a KM of 41 microM. The previously reported catalysis of GSB has a k(cat) of 0.00078 min(-1) and KM of 5 microM. The k(cat) for A216H-catalyzed hydrolysis of GS-2 is thus 4.1 times higher than for GSB. The catalytic proficiency (k(cat)/KM)/k(uncat) for GS-2 is 3 x 10(6) M(-1). The promiscuous feature of the wt protein towards a range of different substrates has not been conserved in A216H but we have obtained a selective enzyme with high demands on the substrate.

Catalysis↗
PubMed1976

Esterolytic activities of rat intestinal mucosa. 1. Characterization, cellular distribution and subcellular localization of a glycerol-ester hydrolase.

The preferential cellular distribution in the villus tip and the subcellular localization in the endoplasmic reticulum of an intestinal glycerol-ester hydrolase from rat mucosa are described. The enzyme is shown not to be from either pancreatic or bacterial origin; it catalyzes the hydrolysis of short- and medium chain triglycerides and of p-nitrophenylacetate. Contrarily to the specificity found for the pig intestinal lipase (Serrero, Négrel and Ailhaud, 1975), no activity is detectable against acylCoA; a thiolester hydrolase different from the glycerol-ester hydrolase was demonstrated after differential solubilization and chromatographic separation. A high proportion of glycerol-ester hydrolase is present in the intestinal lumen; its possible complementary role in lipid degradation is discussed.

Aging↗
PubMed1986

New regional localisations for HAGH and PGP on human chromosome 16.

The chromosomal locations for the electrophoretic markers hydroxyacyl glutathione hydrolase (HAGH) and phosphoglycolate phosphatase (PGP) were examined using a human-mouse hybrid panel of chromosome 16. The assignment for HAGH was confirmed to chromosome 16 using a cell line with chromosome 16 as the only human chromosome. Both HAGH and PGP were present only in cell lines containing human 16p13. This localisation for PGP indirectly places the tightly linked genes for the alpha-globin cluster and adult polycystic kidney disease on 16p13.

Animals↗
PubMed1981

Isolation of palmitoyl-CoA hydrolases from human blood platelets.

The palmitoyl-CoA hydrolase activity, which in human blood platelets is mainly localized in the cytosol fraction [Berge, Vollset & Farstad (1980) Scand. J. Clin. Lab. Invest. 40, 271--279], was found to be extremely labile. Inclusion of glycerol or palmitoyl-CoA stabilized the activity during preparation. Gel-filtration studies revealed multiple forms of the enzyme with molecular weights corresponding to about 70 000, 40 000 and 24 000. The relative recovery of the mol.wt.-70 000 form was increased by the presence of 20% (v/v) glycerol or 10 microM-palmitoyl-CoA. The three enzyme forms are probably unrelated, since they were not interconvertible. The three different species of palmitoyl-CoA hydrolase were purified by DEAE-cellulose and hydroxyapatite chromatography, isoelectric focusing and high-pressure liquid chromatography (h.p.l.c.) to apparent homogeneity. The three enzymes had isoelectric points (pI) of 7.0, 6.1 and 4.9. The corresponding molecular weights were 27 000--33 000, 66 000--72 000 and 45 000--49 000, calculated from h.p.l.c. and Ultrogel AcA-44 chromatography. The apparently purified enzymes were unstable, as most of the activity was lost during purification. The enzyme with an apparent molecular weight of 45 000--49 000 was split into fractions with molecular weights of less than 10 000 by re-chromatography on h.p.l.c. concomitantly with a loss of activity. The stimulation of the activity by the presence of serum albumin seems to depend on the availability of palmitoyl-CoA, as has been reported for other palmitoyl-CoA hydrolases. [Berge & Farstad (1979) Eur. J. Biochem. 96, 393--401].

Blood Platelets↗
PubMed1983

An enzymic cycling method for the determination of free fatty acids with acyl-CoA synthetase and acyl-CoA hydrolase.

A method for measuring free fatty acids by enzymic cycling is described. Free fatty acids are converted to acyl-CoAs by acyl-CoA synthetase, then the acyl-CoAs are hydrolyzed back to the free fatty acids by acyl-CoA hydrolase in a cyclic fashion. The amounts of AMP produced during this cyclic reaction are determined from the absorbance at 340 nm in the presence of AMP deaminase and glutamate dehydrogenase. This method is sensitive to as low as 0.1 nmol of free fatty acids, and the standard curve is linear up to 1.0 nmol. This method shows a broad specificity for long-chain fatty acids (C12--C20) and the recoveries of fatty acids added to bacterial cell-free extracts are more than 90%.

AMP Deaminase↗
PubMed1990

Localisation of human PGP and HAGH genes to 16p13.3.

Gene loci for phosphoglycolate phosphatase and hydroxyacyl glutathione hydrolase were localized within human chromosome band 16p13.3. This result was determined by the electrophoretic detection of enzymes from a human x mouse somatic cell panel, the members of which carried portions of human chromosome 16 with precisely defined breakpoints.

Chromosome Mapping↗
PubMed1983

Differential effects of altered hormonal state on the induction of acyl-CoA hydrolases and peroxisomal beta-oxidation by clofibric acid.

Induction of hydrolase I, hydrolase II, peroxisomal beta-oxidation and hepatomegaly caused by clofibric acid (rho-chlorophenoxyisobutyric acid) administration was investigated in relation to alterations in hormonal state of glucocorticoid, thyroid hormone and insulin. (1) In adrenalectomized rats, the ability to induce hydrolase I was depressed effectively and little hepatomegaly was produced. Hydrolase II and peroxisomal beta-oxidation were induced to a similar extent, compared to those of intact rats. (2) In hypothyroid rats, induction of hydrolase I, hydrolase II, peroxisomal beta-oxidation and hepatomegaly was reduced. In hyperthyroid rats, the ability to induce hydrolase I, hydrolase II and peroxisomal beta-oxidation was depressed, although hepatomegaly was produced by the same or a greater extent as in intact rats. (3) In diabetic rats, marked reduction of ability to induce both hydrolase I and II was observed and induction of hepatomegaly was depressed slightly, although peroxisomal beta-oxidation was induced normally. Differences in the response of four parameters (hydrolase I, hydrolase II, peroxisomal beta-oxidation and liver size) to alterations in hormonal state suggest that the four biological responses to clofibric acid may each be mediated through distinct mechanism(s) other and regulated by distinct hormone(s).

Adrenalectomy↗
PubMed1985

Cardiac and hepatic acyl-CoA and acylcarnitine hydrolase activities in clofibrate-fed rabbits.

Catalase activity in the heart of male rabbits was 21% of that found in the liver; clofibrate feeding (0.3% w/w for 10 days) resulted in an 80% increase in both cardiac and hepatic catalase activities. Fatty acyl-CoA oxidase activity in control heart was 11% of that found in control liver; this peroxisomal activity did not increase subsequent to clofibrate feeding. Only acyl-CoA hydrolase activity in the cardiac supernatant was elevated by clofibrate feeding. Acylcarnitine hydrolase activity was increased significantly in the homogenate, extract and supernatant of both heart and liver from the clofibrate-fed rabbit. Clofibrate feeding increased CoASH and carnitine tissue levels in heart and liver.

Animals↗
PubMed1989

Effects of experimental hypo- and hyperthyroidism on hepatic long-chain fatty acyl-CoA synthetase and hydrolase.

The effects of T3 treatment and thyroidectomy on rat liver microsomal long-chain fatty acyl-CoA (LCFA-CoA) synthetase and LCFA-CoA hydrolase activities were determined. Hyperthyroid rats had a 36-42% decrease in LCFA-CoA synthetase with no change in hydrolase activity. This may contribute to the redirection of fatty acids from esterification to oxidation reactions in hyperthyroidism. Thyroidectomized rats had a 40-44% decrease in synthetase and a 27-42% decrease in LCFA-CoA hydrolase activity. The decrease in both LCFA-CoA synthetase and hydrolase activities in hypothyroidism may indicate that the LCFA-CoA turnover in this futile cycle is decreased in the liver.

Animals↗
PubMed1979

Dual localization of long-chain acyl-CoA hydrolase in rat liver: one in the microsomes and one in the mitochondrial matrix.

Subcellular fractionation studies of rat liver localized the activity of palmitoyl-L-carnitine hydrolase to the microsomal fraction whereas palmitoyl-CoA hydrolase activity was found both in the microsomal fraction and in mitochrondria. An unusual biphasic sataration curve for palmitoyl-CoA was observed when intact mitochondrial hydrolase activity. Disruption of the mitochondrial structure doubled the palmitoyl-CoA hydrolysis. Discontinuous sucrose gradient centrifugation and digitonin fractionation of rat liver mitochondria demonstrated that a palmitoyl-CoA hydrolase was associated with the matrix fraction. Pure matrix and microsomal fractions showed that the two hydrolase activities were differently affected by the presence of divalent cations. Both the specific activity and the saturation concentration of palmitoyl-CoA were higher for the microsomal enzyme than for the matrix-associated enzyme.

Animals↗
PubMed1986

Genetic and biochemical characterization of an Escherichia coli K-12 mutant deficient in acyl-coenzyme A thioesterase II.

Mutants of Escherichia coli deficient in thioesterase II activity were isolated by taking advantage of the fact that thioesterase I specifically hydrolyzes long-chain (C12 to C18) acyl coenzyme A (CoA) esters but is unable to cleave the short-chain substrate decanoyl-CoA. One of these lesions (designated tesB1) reduces thioesterase II activity to about 10% of the normal level. The mutant enzyme activity was abnormally labile to temperature, but it was normal in all the other characteristics examined (pH optimum, Km for decanoyl-CoA, molecular weight). The level of thioesterase I activity was unaffected by the tesB1 lesion. The tesB locus was mapped with a closely linked Tn10 insertion. tesB was mapped to minute 10 of the E. coli linkage map, close to the lon locus. The clockwise gene order is lon tesB acrA dnaZ. The tesB mutation is recessive. We found no phenotype for the mutation. The fatty acid compositions of the phospholipids, lipid A, and lipoprotein components are normal in thioesterase II mutants. These data show that thioesterases I and II of E. coli are encoded by different genetic loci and strongly suggest that tesB is the structural gene for thioesterase II.

Chromosome Mapping↗
PubMed1999

Characterization of a human MHC class III region gene product with S-thioesterase activity.

Palmitoylated proteins contain a 16-carbon saturated fatty acyl group that is post-translationally attached by a labile thioester bond. These modified proteins are mainly membrane-bound; the lability of the thioester bond allows the process to be reversible, a unique property of this modification. We report here that the gene for G14, located in the class III region of the human MHC, encodes a polypeptide with significant sequence similarity to mammalian palmitoyl protein thioesterase (PPT1), an enzyme that removes palmitate from palmitoylated proteins. The gene for G14, also known as PPT2, is transcribed as at least five different transcripts, which are expressed in different cell lines of the immune system. Immunoprecipitation of these mammalian cells, with an anti-G14 antiserum, showed a specific band of approx. 42 kDa in cell extracts and supernatants. Expression of the G14 cDNA in the baculovirus system revealed that it encoded a secreted glycosylated polypeptide with S-thioesterase activity. The enzymic activity of the recombinant G14 protein was further characterized in quantitative spectrophotometric assays, which revealed that it had the highest S-thioesterase activity for the acyl groups palmitic and myristic acid followed by other long-chain acyl substrates. The S-thioesterase activity of the G14 protein was found to be considerably higher in supernatants than in cell extracts, which was consistent with the protein's being secreted. The G14 polypeptide contains, in addition to an N-terminal lipase domain, a C-terminal domain common to the cytokine receptor superfamily, which might determine the substrate specificity and/or the protein target of the G14 protein.

Amino Acid Sequence↗
PubMed1984

Generation of fatty acids by an acyl esterase in the bioluminescent system of Photobacterium phosphoreum.

The fatty acid reductase complex from Photobacterium phosphoreum has been discovered to have a long chain ester hydrolase activity associated with the 34K protein component of the complex. This protein has been resolved from the other components (50K and 58K) of the fatty acid reductase complex with a purity of greater than 95% and found to catalyze the transfer of acyl groups from acyl-CoA primarily to thiol acceptors with a low level of transfer to glycerol and water. Addition of the 50K protein of the complex caused a dramatic change in specificity increasing the transfer to oxygen acceptors. The acyl-CoA hydrolase activity increased almost 10-fold, and hence free fatty acids can be generated by the 34K protein when it is present in the fatty acid reductase complex. Hydrolysis of acyl-S-mercaptoethanol and acyl-1-glycerol and the ATP-dependent reduction of the released fatty acids to aldehyde for the luminescent reaction were also demonstrated for the reconstituted fatty acid reductase complex, raising the possibility that the immediate source of fatty acids for this reaction in vivo could be the membrane lipids and/or the fatty acid synthetase system.

Aldehyde Oxidoreductases↗
PubMed1990

In vitro study of docosahexaenoic acid incorporation into phosphatidylcholine by enzymes of rat heart.

Several studies have shown that in animals fed fish oils, docosahexaenoic acid (DHA) is incorporated into cardiac phosphatidylcholines (PC) mainly at the expense of arachidonic acid. In this study we were interested in examining if the enzymatic system involved in the remodeling of membrane PC presented any selectivity for DHA in rat heart. The enzymes that were studied from sequential incubations carried out in parallel, were acyl-CoA synthetase (EC 6.2.1.3) and acyl-CoA:lysophosphatidylcholine acyltransferase (EC 2.3.1.23) (ACLAT). The heart preparations examined were homogenates of whole heart and of purified cultured rat ventricular myocytes. Results showed that ACLAT tended to preferentially incorporate into PC the polyunsaturated fatty acids of the n-6 series (+30%) rather than those of the n-3 series. DHA, however, inhibited the incorporation of arachidonic acid (AA) into PC by 50% at a molar ratio (DHA/AA) of 1.5. This phenomenon seems to be related to the competitive inhibition exerted by DHA on the thio-esterification of AA, a reaction catalyzed by acyl-CoA synthetase. This inhibitory effect appears to be dependent on the kinetic properties of the acyl-CoA synthetase toward DHA which, among the fatty acids examined, exhibited the lowest apparent Km and Vmax. It is suggested that the intracellular pool of DHA-CoA is the determinant species in altering the DHA composition of cardiac PC in animals given fish oils.

Animals↗