|Tovar-Mendez, A - UNIV OF MISSOURI|
|Randall, D - UNIV OF MISSOURI|
Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: August 3, 2002
Publication Date: N/A
Technical Abstract: Pyruvate dehydrogenase kinase (PDK) is the primary regulator of flux through the mitochondrial pyruvate dehydrogenase complex. The PDK is a member of the ATPase/kinase superfamily. Member proteins of this family are characterized by four signature sequences in the catalytic domain (N-, D-, F-, and G-boxes), and include ATPases, GyrB, HSP90, and protein His-kinases (PHKs). Contrary to HPKs, PDK is a protein Ser-kinase, and does not employ a phospho-His intermediate. Results from mutagenesis of rat PDK2 suggest an ATPase-like mechanism (Tuganova et al. (2001) J. Biol. Chem. 276:17994-17999). It was proposed that a conserved Glu residue within the N-box of PDK is a general base catalyst for phospho-transfer, and that a neighboring His residue polarizes the Glu. However, the Glu-polarizing His residue is not present in plant PDK sequences. Rather, plant PDK sequences have a Leu residue in this position (Leu234 in the Arabidopsis thaliana PDK sequence). Mutation of the polarizing His in rat PDK2 reduced activity to 10% of the rate seen for the wild-type recombinant protein. To test the polarizing His residue hypothesis, we prepared the L234H mutant of AtPDK. However, this mutant had the same activity as the wild-type enzyme. This result is inconsistent with the polarizing-His model. However, the AtPDK sequence includes a His residue one position upstream of L234, and we are constructing the H233A mutant to test for a role in Glu-polarization in plant PDKs.