Crystallographic and kinetic studies of human mitochondrial acetoacetyl-CoA thiolase: the importance of potassium and chloride ions for its structure and function.
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.
Mesh Terms:
Acetyl-CoA C-Acetyltransferase, Acyl Coenzyme A, Amino Acid Sequence, Apoenzymes, Binding Sites, Catalysis, Chlorides, Conserved Sequence, Crystallography, X-Ray, Dimerization, Dipeptides, Escherichia coli, Humans, Hydrogen Bonding, Ions, Kinetics, Mitochondria, Models, Molecular, Molecular Sequence Data, Phenylalanine, Potassium, Proline, Protein Binding, Protein Structure, Quaternary, Protein Structure, Secondary, Protein Structure, Tertiary, Protein Subunits, Recombinant Proteins, Sequence Homology, Amino Acid, Substrate Specificity
Acetyl-CoA C-Acetyltransferase, Acyl Coenzyme A, Amino Acid Sequence, Apoenzymes, Binding Sites, Catalysis, Chlorides, Conserved Sequence, Crystallography, X-Ray, Dimerization, Dipeptides, Escherichia coli, Humans, Hydrogen Bonding, Ions, Kinetics, Mitochondria, Models, Molecular, Molecular Sequence Data, Phenylalanine, Potassium, Proline, Protein Binding, Protein Structure, Quaternary, Protein Structure, Secondary, Protein Structure, Tertiary, Protein Subunits, Recombinant Proteins, Sequence Homology, Amino Acid, Substrate Specificity
Biochemistry
Date: Apr. 10, 2007
PubMed ID: 17371050
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