title: 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.
authors: Biocenter Oulu and Department of Biochemistry, University of Oulu, P.O. Box 3000, FIN-90014 Oulu, Finland.
authors: Antti M
authors: AM
authors: Haapalainen
authors: Gitte
authors: G
authors: Meriläinen
authors: Päivi L
authors: PL
authors: Pirilä
authors: Naomi
authors: N
authors: Kondo
authors: Toshiyuki
authors: T
authors: Fukao
authors: Rik K
authors: RK
authors: Wierenga
mesh_terms: D000101:Acetyl-CoA C-Acetyltransferase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000214:Acyl Coenzyme A / Q000378:metabolism; D000595:Amino Acid Sequence; D001051:Apoenzymes / Q000737:chemistry; D001665:Binding Sites; D002384:Catalysis; D002712:Chlorides / Q000737:chemistry / Q000378:metabolism*; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D019281:Dimerization; D004151:Dipeptides / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D006860:Hydrogen Bonding; D007477:Ions; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010649:Phenylalanine / Q000737:chemistry; D011188:Potassium / Q000737:chemistry / Q000378:metabolism*; D011392:Proline / Q000737:chemistry; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D000214:Acyl Coenzyme A; D001051:Apoenzymes; D002712:Chlorides; D004151:Dipeptides; D007477:Ions; D021122:Protein Subunits; D011994:Recombinant Proteins; C010667:acetoacetyl CoA; D010649:Phenylalanine; C037766:2-methylacetoacetyl-coenzyme A; D011392:Proline; D000101:Acetyl-CoA C-Acetyltransferase; D011188:Potassium