title: Roles of active site and novel K+ ion-binding site residues in human mitochondrial branched-chain alpha-ketoacid decarboxylase/dehydrogenase.
abstract: The human mitochondrial branched-chain alpha-ketoacid decarboxylase/dehydrogenase (BCKD) is a heterotetrameric (alpha(2)beta(2)) thiamine diphosphate (TDP)-dependent enzyme. The recently solved human BCKD structure at 2.7 A showed that the two TDP-binding pockets are located at the interfaces between alpha and beta' subunits and between alpha' and beta subunits. In the present study, we show that the E76A-beta' mutation results in complete inactivation of BCKD. The result supports the catalytic role of the invariant Glu-76-beta' residue in increasing basicity of the N-4' amino group during the proton abstraction from the C-2 atom on the thiazolium ring. A substitution of His-146-beta' with Ala also renders the enzyme completely inactive. The data are consistent with binding of the alpha-ketoacid substrate by this residue based on the Pseudomonas BCKD structure. Alterations in Asn-222-alpha, Tyr-224-alpha, or Glu-193-alpha, which coordinates to the Mg(2+) ion, result in an inactive enzyme (E193A-alpha) or a mutant BCKD with markedly higher K(m) for TDP and a reduced level of the bound cofactor (Y224A-alpha and N222S-alpha). Arg-114-alpha, Arg-220-alpha, and His-291-alpha interact with TDP by directly binding to phosphate oxygens of the cofactor. We show that natural mutations of these residues in maple syrup urine disease (MSUD) patients (R114W-alpha and R220W-alpha) or site-directed mutagenesis (H291A-alpha) also result in an inactive or partially active enzyme, respectively. Another MSUD mutation (T166M-alpha), which affects one of the residues that coordinate to the K(+) ion on the alpha subunit, also causes inactivation of the enzyme and an attenuated ability to bind TDP. In addition, fluorescence measurements establish that Trp-136-beta in human BCKD is the residue quenched by TDP binding. Thus, our results define the functional roles of key amino acid residues in human BCKD and provide a structural basis for MSUD.
authors.affiliation: Departments of Biochemistry and Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.
authors.forename: R M
authors.initials: RM
authors.lastname: Wynn
authors.forename: R
authors.initials: R
authors.lastname: Ho
authors.forename: J L
authors.initials: JL
authors.lastname: Chuang
authors.forename: D T
authors.initials: DT
authors.lastname: Chuang
mesh_terms: D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); D001665:Binding Sites; D020134:Catalytic Domain; D006801:Humans; D007658:Ketone Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008928:Mitochondria / Q000201:enzymology*; D008958:Models, Molecular; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016296:Mutagenesis; D010100:Oxygen / Q000378:metabolism; D011188:Potassium / Q000378:metabolism*; D011487:Protein Conformation
chemical_list: D009097:Multienzyme Complexes; D007658:Ketone Oxidoreductases; D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); D011188:Potassium; D010100:Oxygen