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Papers In Press, published online ahead of print May 27, 2004
Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX 75390-9038
Corresponding Author: david.chuang{at}UTSouthwestern.edu
The decarboxylase/dehydrogenase (E1b) component of the 4-megadalton human branched-chain a-ketoacid dehydrogenase (BCKD) metabolic machine is a thiamin diphosphate (ThDP)-dependent enzyme with a heterotetrameric cofactor-binding fold. The E1b component catalyzes the decarboxylation of a-ketoacids and the subsequent reductive acylation of the lipoic acid-bearing domain (LBD) from the 24-meric transacylase (E2b) core. In the present study, we show that the binding of cofactor ThDP to the E1b active site induces a disorder-to-order transition of the conserved phosphorylation loop carrying the two phosphorylation sites Ser292-a and Ser302-a, as deduced from the 1.80 –1.85 Å apo- and holo-E1b structures. The induced loop conformation is essential for the recognition of lipoylated LBD to initiate E1b-catalyzed reductive acylation. Alterations of invariant Arg287-a, Asp295-a, Tyr300-a and Arg301-a that form a hydrogen-bonding network in the phosphorylation loop result in the disordering of the loop conformation, accompanied by the impaired binding and diminished reductive acylation of lipoylated LBD. In contrast, kcat values for decarboxylation of the a-ketoacid are higher in these E1b mutants than in wild-type E1b, with higher Km for the substrate in the former. ThDP binding that orders the loop prevents phosphorylation of E1b by the BCKD kinase, and averts the inactivation of wild-type E1b, but not the above mutants, by this covalent modification. Our results establish that the crosstalk between the bound ThDP and the phosphorylation loop conformation serves as a feed-forward switch for multiple reaction steps in the BCKD metabolic machine.
J. Biol. Chem, 10.1074/jbc.M403611200
Submitted on April 1, 2004
Revised on May 24, 2004
Accepted on May 26, 2004
Crosstalk between thiamin diphosphate binding and phosphorylation loop conformation in human branched-chain a-ketoacid decarboxylase/dehydrogenase
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