Roles of Active Site and Novel K⁺ Ion-binding Site Residues in Human Mitochondrial Branched-chain α-Ketoacid Decarboxylase/Dehydrogenase

The human mitochondrial branched-chain α-ketoacid decarboxylase/dehydrogenase (BCKD) is a heterotetrameric (α ₂β₂) thiamine diphosphate (TDP)-dependent enzyme. The recently solved human BCKD structure at 2.7 Å showed that the two TDP-binding pockets are located at the interfaces between α and β' subunits and between α' and β subunits. In the present study, we show that the E76A-β' mutation results in complete inactivation of BCKD. The result supports the catalytic role of the invariant Glu-76-β' 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-β' with Ala also renders the enzyme completely inactive. The data are consistent with binding of the α-ketoacid substrate by this residue based on the Pseudomonas BCKD structure. Alterations in Asn-222-α, Tyr-224-α, or Glu-193-α, which coordinates to the Mg²⁺ ion, result in an inactive enzyme (E193A-α) or a mutant BCKD with markedly higher K_m for TDP and a reduced level of the bound cofactor (Y224A-α and N222S-α). Arg-114-α, Arg-220-α, and His-291-α 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-α and R220W-α) or site-directed mutagenesis (H291A-α) also result in an inactive or partially active enzyme, respectively. Another MSUD mutation (T166M-α), which affects one of the residues that coordinate to the K⁺ ion on the α subunit, also causes inactivation of the enzyme and an attenuated ability to bind TDP. In addition, fluorescence measurements establish that Trp-136-β 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.