Roles of Active Site and Novel K+ Ion-binding Site Residues in Human Mitochondrial Branched-chain α-Ketoacid Decarboxylase/Dehydrogenase*
Roles of Active Site and Novel K+ Ion-binding Site Residues in Human Mitochondrial Branched-chain α-Ketoacid Decarboxylase/Dehydrogenase*
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人线粒体支链 α-酮酸脱羧酶/脱氢酶中活性位点和新型 K+ 离子结合位点残基的作用*
DOI：
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发表时间：
2001
影响因子：
4.8
通讯作者：
D. Chuang
中科院分区：
文献类型：
--
作者：
R. Wynn;Roxanne Ho;J. Chuang;D. Chuang
The human mitochondrial branched-chain α-ketoacid decarboxylase/dehydrogenase (BCKD) is a heterotetrameric (α2β2) 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.
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单价阳离子和无机磷酸盐改变支链 α-酮酸脱氢酶激酶活性和抑制剂敏感性。
影响因子：
3.9
作者：
Shimomura,Y;Kuntz,MJ;Suzuki,M;Ozawa,T;Harris,RA
通讯作者：
Harris,RA
影响因子：
2.9
作者：
DYDA, F;FUREY, W;JORDAN, F
通讯作者：
JORDAN, F
影响因子：
2.9
作者：
Hasson, MS;Muscate, A;Ringe, D
通讯作者：
Ringe, D
支链α-酮酸脱氢酶复合物磷酸化位点的定点诱变。
DOI：
--
发表时间：
1994
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Zhao,Y;Hawes,J;Popov,KM;Jaskiewicz,J;Shimomura,Y;Crabb,DW;Harris,RA
通讯作者：
Harris,RA