Submitted to: Meeting Abstract
Publication Type: Abstract only
Publication Acceptance Date: 4/12/2000
Publication Date: N/A
Citation: Interpretive Summary:
Technical Abstract: Pyruvate dehydrongenase kinase (PDK) is the primary regulator of flux through the mitochondrial pyruvate dehydrogenase complex (PDC). PDK inactivates PDC by multi-site phosphorylation of specific Ser residues in the E1 alpha subunit of PDC. Reactivation is then catalyzed by a specific intrinsic phospho-E1 phosphatase. Analysis of cloned cDNAs revealed that the PDK-deduced amino acid sequences lack typical eukaryotic Ser/Thr kinas domains; instead they contain the five signature domains of prokaryotic, two-component, protein histidine kinases. Unlike Ser/Thr kinases, protein histidine kinases autophosphorylate a conserved His residue followed by phosphotransfer to an Asp residue within the response-regulator domain or protein. Thus, PDKs have a protein His kinase structure with Ser kinase activity. To better understand these unique protein kinases, we have begun molecular analysis of the reaction mechanism. We have identified PDK cDNAs from Zea mays and Arabidopsis thaliana. The cloned kinases were expressed in and purified from Escherichia coli. Purified, recombinant A. thaliana PDK was fully active; capable of inactivating kinase-depleted pea mitochondrial PDC by Ser phosphorylation. Treatment of recombinant A. thaliana PDK with the His-directed reagents diethyl pyrocarbonate (DEPC) or dichloro (2,2':6',2"-terpydridine)-platinum (II) dihydrate resulted in a marked inhibition of autophosphorylation. In addition, DEPC treatment abolished the ability of PDK to trans-phosphorylate and inactivate PDC. These results validate the sequence-based prediction that PDKs require His residues for activity. To further study the PDK reaction mechanism, we performed site-directed mutagenesis of a His residue purported to be involved in phosphotransfer.