Mutation of residues 423 (Met/Ile), 444 (Thr/Met), and 506 (Asn/Ser) confer cholesteryl esterase activity on rat lung carboxylesterase - Ser-506 is required for activation by cAMP-dependent protein kinase

Department of Biochemistry and Molecular Biology, Virginia Commonwealth University, Ричмонд, Virginia, United States
Journal of Biological Chemistry (Impact Factor: 4.57). 08/2001; 276(35):33165-74. DOI: 10.1074/jbc.M105644200
Source: PubMed


Site-directed mutagenesis is used to identify amino acid residues that dictate reported differences in substrate specificity
between rat hepatic neutral cytosolic cholesteryl ester hydrolase (hncCEH) and rat lung carboxylesterase (LCE), proteins differing
by only 4 residues in their primary sequences. Beginning with LCE, the substitution Met423 → Ile423alone or in combination with other mutations increased activity withp-nitrophenylcaprylate (PNPC) relative to more hydrophilicp-nitrophenylacetate (PNPA), typical of hncCEH. The substitution Thr444 → Met444 was necessary but not sufficient for expression of cholesteryl esterase activity in COS-7 cells. The substitution Asn506 → Ser506, creating a potential phosphorylation site, uniformly increased activity with both PNPA and PNPC, was necessary but not sufficient
for expression of cholesteryl esterase activity and conferred susceptibility to activation by cAMP-dependent protein kinase,
a property of hncCEH. The 3 mutations in combination were necessary and sufficient for expression of cholesteryl esterase
activity by the mutated LCE. The substitution Gln186 → Arg186 selectively reduced esterase activity with PNPA and PNPC but was not required for cholesteryl esterase activity. Homology
modeling from x-ray structures of acetylcholinesterases is used to propose three-dimensional models for hncCEH and LCE that
provide insight into the effects of these mutations on substrate specificity.

Full-text preview

Available from:
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Hormone-sensitive lipase (HSL) is expressed predominantly in white and brown adipose tissue where it is believed to play a crucial role in the lipolysis of stored triglycerides (TG), thereby providing the body with energy substrate in the form of free fatty acids (FFA). From in vitro assays, HSL is known to hydrolyze TG, diglycerides (DG), cholesteryl esters, and retinyl esters. In the current study we have generated HSL knock-out mice and demonstrate three lines of evidence that HSL is instrumental in the catabolism of DG in vivo. First, HSL deficiency in mice causes the accumulation of DG in white adipose tissue, brown adipose tissue, skeletal muscle, cardiac muscle, and testis. Second, when tissue extracts were used in an in vitro lipase assay, a reduced FFA release and the accumulation of DG was observed in HSL knock-out mice which did not occur when tissue extracts from control mice were used. Third, in vitro lipolysis experiments with HSL-deficient fat pads demonstrated that the isoproterenol-stimulated release of FFA was decreased and DG accumulated intracellularly resulting in the essential absence of the isoproterenol-stimulated glycerol formation typically observed in control fat pads. Additionally, the absence of HSL in white adipose tissue caused a shift of the fatty acid composition of the TG moiety toward increased long chain fatty acids implying a substrate specificity of the enzymein vivo. From these in vivo results we conclude that HSL is the rate-limiting enzyme for the cellular catabolism of DG in adipose tissue and muscle.
    Full-text · Article · Mar 2002 · Journal of Biological Chemistry
  • [Show abstract] [Hide abstract]
    ABSTRACT: Mammalian carboxylesterases cleave the anticancer prodrug CPT-11 (Irinotecan) into SN-38, a potent topoisomerase I poison, and 4-piperidino-piperidine (4PP). We present the 2.5 A crystal structure of rabbit liver carboxylesterase (rCE), the most efficient enzyme known to activate CPT-11 in this manner, in complex with the leaving group 4PP. 4PP is observed bound adjacent to a high-mannose Asn-linked glycosylation site on the surface of rCE. This product-binding site is separated from the catalytic gorge by a thin wall of amino acid side chains, suggesting that 4PP may be released through this secondary product exit pore. The crystallographic observation of a leaving group bound on the surface of rCE supports the 'back door' product exit site proposed for the acetylcholinesterases. These results may facilitate the design of improved anticancer drugs or enzymes for use in viral-directed cancer cotherapies.
    No preview · Article · Jun 2002 · Nature Structural Biology
  • [Show abstract] [Hide abstract]
    ABSTRACT: Phospholipase A(2) (PLA(2)) was purified to homogeneity from the supernatant fraction of rat testis homogenate. The purified 63-kDa enzyme did not require Ca(2+) ions for activity and exhibited both phosphatidic acid-preferring PLA(2) and monoacylglycerol lipase activities with a modest specificity toward unsaturated acyl chains. Anionic detergents enhanced these activities. Serine-modifying irreversible inhibitors, (p-amidinophenyl) methanesulfonyl fluoride and methylarachidonyl fluorophosphonate, inhibited both activities to a similar extent, indicating a single active site is involved in PLA(2) and lipase activities. The sequence of NH(2)-terminal 12 amino acids of purified enzyme was identical to that of a carboxylesterase from rat liver. The optimal pH for PLA(2) activity (around 5.5) differed from that for lipase activity (around 8.0). At pH 5.5 the enzyme also hydrolyzed bis(monoacylglycerol) phosphate, or lysobisphosphatidic acid (LBPA), that has been hitherto known as a secretory PLA(2)-resistant phospholipid and a late endosome marker. LBPA-enriched fractions were prepared from liver lysosome fractions of chloroquine-treated rats, treated with excess of pancreatic PLA(2), and then used for assaying LBPA-hydrolyzing activity. LBPA and the reaction products were identified by microbore normal phase high performance liquid chromatography/electrospray ionization ion-trap mass spectrometry. These enzymatic properties suggest that the enzyme can metabolize phosphatidic and lysobisphosphatidic acids in cellular acidic compartments.
    No preview · Article · Dec 2002 · Journal of Biological Chemistry
Show more

We use cookies to give you the best possible experience on ResearchGate. Read our cookies policy to learn more.