Crystallographic and Kinetic Studies of Human Mitochondrial Acetoacetyl-CoA Thiolase:  The Importance of Potassium and Chloride Ions for Its Structure and Function^,

Abstract

Thiolases are CoA-dependent enzymes which catalyze the formation of a carbon-carbon bond in a Claisen condensation step and its reverse reaction via a thiolytic degradation mechanism. Mitochondrial acetoacetyl-coenzyme A (CoA) thiolase (T2) is important in the pathways for the synthesis and degradation of ketone bodies as well as for the degradation of 2-methylacetoacetyl-CoA. Human T2 deficiency has been identified in more than 60 patients. A unique property of T2 is its activation by potassium ions. High-resolution human T2 crystal structures are reported for the apo form and the CoA complex, with and without a bound potassium ion. The potassium ion is bound near the CoA binding site and the catalytic site. Binding of the potassium ion at this low-affinity binding site causes the rigidification of a CoA binding loop and an active site loop. Unexpectedly, a high-affinity binding site for a chloride ion has also been identified. The chloride ion is copurified, and its binding site is at the dimer interface, near two catalytic loops. A unique property of T2 is its ability to use 2-methyl-branched acetoacetyl-CoA as a substrate, whereas the other structurally characterized thiolases cannot utilize the 2-methylated compounds. The kinetic measurements show that T2 can degrade acetoacetyl-CoA and 2-methylacetoacetyl-CoA with similar catalytic efficiencies. For both substrates, the turnover numbers increase approximately 3-fold when the potassium ion concentration is increased from 0 to 40 mM KCl. The structural analysis of the active site of T2 indicates that the Phe325-Pro326 dipeptide near the catalytic cavity is responsible for the exclusive 2-methyl-branched substrate specificity.

Cloning and Characterization of Mevalonate Pathway Genes in a Natural Rubber Producing Plant,Hevea brasiliensis

“…In addition, the AACTs in H. brasiliensis showed 76%-86% identity to those in A. thaliana and O. sativa, and 53-55% identity to that in yeast. Moreover, the AACT sequences in H. brasiliensis also had amino acid residues crucial to the thiolase activity of acetoacetylCoA thiolase 15) that were well conserved ( Fig. 2A).…”

Quantification of Mitochondrial Acetylation Dynamics Highlights Prominent Sites of Metabolic Regulation

“…Biochemical Assessment of Acat1 Activity-Recombinant Acat1 (amino acids 31-424) with a C-terminal hexahistidine tag was overexpressed in Escherichia coli and purified using metal affinity resin based on previous methods (24,25). For site-specific acetyllysine incorporation, an acetyl-lysyl-tRNA synthetase/tRNA CUA pair that recognizes an amber codon was co-expressed with Acat1 in the presence of 10 mM acetyllysine and 20 mM nicotinamide (26 -28).…”

“…Acat1 Activity Assays-Activity was assessed as described previously (24,30). Briefly, the reaction mixture contained 50 mM Tris-HCl (pH 8.1), 20 mM MgCl 2 , 10 M acetoacetyl-CoA, 40 mM KCl, and 8 ng of purified Acat1 enzyme (diluted into a buffer of 50 mM HEPES (pH 6.6), 9.5% glycerol, and 0.5 mg/ml gelatin).…”

“…Electrostatic Modeling-To assess the changes in charge complementarity for CoA in the active site of Acat1 caused by lysine acetylation, the tetrameric coordinates of Protein Data Bank code 2IBW (24) were prepared for electrostatic calculations. Water and other ligands were removed from the model.…”

Protein-Protein Interactions in the β-Oxidation Part of the Phenylacetate Utilization Pathway

“…In animal cells fatty acid ␤-oxidation takes place in mitochondria and peroxisomes. In mitochondria the oxidation steps may be performed by monofunctional enzymes: ECH (17, 18), 3-hydroxyacyl-CoA dehydrogenase (19), and 3-ketoacyl-CoA thiolase (20), or by a trifunctional membrane-bound enzyme complex similar to the bacterial FOM (21). In peroxisomes the multifunctional enzymes, similar to the ␣-subunit of the bacterial FOM, catalyze 2-enoyl-CoA hydration and 3-hydroxyacyl-CoA dehydrogenation (22), whereas 3-ketoacylCoA thiolase is a separate monofunctional enzyme (23).…”