Roles of active site and novel K+ ion-binding site residues in human mitochondrial branched-chain alpha-ketoacid decarboxylase/dehydrogenase

The human mitochondrial branched-chain alpha -ketoacid decarboxylase/dehydr ogenase (BCKD) is a heterotetrameric (alpha (2)beta (2)) thiamine diphospha te (TDP)-dependent enzyme. The recently solved human BCKD structure at 2.7 Angstrom showed that the two TDP-binding pockets are located at the interfa ces between alpha and beta' subunits and between alpha' and beta subunits. In the present study, we show that the E76A-beta' mutation results in compl ete inactivation of BCKD. The result supports the catalytic role of the inv ariant 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 s ubstitution of His-146-beta' with Ala also renders the enzyme completely in active. The data are consistent with binding of the alpha -ketoacid substra te 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 ma rkedly 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 inte ract 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, respec tively Another MSUD mutation (T166M-alpha), which affects one of the residu es that coordinate to the K+ ion on the alpha subunit, also causes inactiva tion of the enzyme and an attenuated ability to bind TDP. In addition, fluo rescence measurements establish that Trp-136-beta in human BCKD is the resi due 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.