Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 276, Issue 6, Pages 4168-4174Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M008038200
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- NIDDK NIH HHS [DK-26758] Funding Source: Medline
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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 Angstrom 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 Mg2+ 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.
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