Abstract
Long-chain acyl-CoA thioesterases hydrolyze long-chain acyl-CoAs to the corresponding free fatty acid and CoASH and may therefore play important roles in regulation of lipid metabolism. We have recently cloned four members of a highly conserved acyl-CoA thioesterase multigene family expressed in cytosol (CTE-I), mitochondria (MTE-I), and peroxisomes (PTE-Ia and -Ib), all of which are regulated via the peroxisome proliferator-activated receptor alpha (Hunt, M. C., Nousiainen, S. E. B., Huttunen, M. K., Orii, K. E., Svensson, L. T., and Alexson, S. E. H. (1999) J. Biol. Chem. 274, 34317-34326). Sequence comparison revealed the presence of putative active-site serine motifs (GXSXG) in all four acyl-CoA thioesterases. In the present study we have expressed CTE-I in Escherichia coli and characterized the recombinant protein with respect to sensitivity to various amino acid reactive compounds. The recombinant CTE-I was inhibited by phenylmethylsulfonyl fluoride and diethyl pyrocarbonate, suggesting the involvement of serine and histidine residues for the activity. Extensive sequence analysis pinpointed Ser(232), Asp(324), and His(358) as the likely components of a catalytic triad, and site-directed mutagenesis verified the importance of these residues for the catalytic activity. A S232C mutant retained about 2% of the wild type activity and incubation with (14)C-palmitoyl-CoA strongly labeled this mutant protein, in contrast to wild-type enzyme, indicating that deacylation of the acyl-enzyme intermediate becomes rate-limiting in this mutant protein. These data are discussed in relation to the structure/function of acyl-CoA thioesterases versus acyltransferases. Furthermore, kinetic characterization of recombinant CTE-I showed that this enzyme appears to be a true acyl-CoA thioesterase being highly specific for C(12)-C(20) acyl-CoAs.
Highlights
Long-chain fatty acids entering cells are rapidly esterified to the corresponding CoA esters prior to degradation via -oxidation in mitochondria or peroxisomes or esterification into triacylglycerol, phospholipids, cholesterol esters, or pro
This paper is available on line at http://www.jbc.org show sequence homology only to a bile acid-CoA:amino acid N-acyltransferase (BAAT), which catalyzes the conjugation of bile acids to glycine or taurine
Previous site-directed mutagenesis experiments of thioesterase II have identified two of the residues that are involved in catalysis: a serine residue found within a so-called esterase consensus sequence, GXSXG [29], and a histidine residue present within a GXH motif [27, 28]
Summary
For kinetic characterization of wild-type and mutant CTE-I, bacteria were solubilized using the BugbusterTM protein extraction kit and Benzonase nuclease (Novagen) and centrifuged for 60 min at 36 000 ϫ g at ϩ4 °C. Acylation and Deacylation of Recombinant Protein—Protein extracts (100 g) of expressed wild-type and mutated CTE-I were incubated with 25 M 14C-palmitoyl-CoA (Sigma) for 1.5 h at 37 °C. Protein loading was quantitated by Western blotting followed by analysis using an Image Master VDS (Amersham Biosciences, Inc.) and Image Master Program, version 3.0. When acyl-CoA thioesterase activity was measured with the serine to cysteine mutant protein, and when the effects of pCMB or DTT were tested on wild-type CTE-I protein, the decrease in absorbance due to cleavage of the thioester bond was followed at 232 nm, and activity was calculated using an ⑀232 nm ϭ 4250 MϪ1 cmϪ1. Protein concentrations were determined according to Bradford [33]
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