Asp295 stabilizes the active-site loop structure of pyruvate dehydrogenase, facilitating phosphorylation of Ser292 by pyruvate dehydrogenase-kinase

Abstract

We have developed an in vitro system for detailed analysis of reversible phosphorylation of the plant mitochondrial pyruvate dehydrogenase complex, comprising recombinant Arabidopsis thaliana α2β2-heterotetrameric pyruvate dehydrogenase (E1) plus A. thaliana E1-kinase (AtPDK). Upon addition of MgATP, Ser292, which is located within the active-site loop structure of E1, is phosphorylated. In addition to Ser292, Asp295 and Gly297 are highly conserved in the E1 active-site loop sequences. Mutation of Asp295 to Ala, Asn, or Leu greatly reduced phosphorylation of Ser292, while mutation of Gly297 had relatively little effect. Quantitative two-hybrid analysis was used to show that mutation of Asp295 did not substantially affect binding of AtPDK to E1. When using pyruvate as a variable substrate, the Asp295 mutant proteins had modest changes in kcat, Km, and kcat/Km values. Therefore, we propose that Asp295 plays an important role in stabilizing the active-site loop structure, facilitating transfer of the γ-phosphate from ATP to the Ser residue at regulatory site one of E1α. © 2011 Tripty A. Hirani et al.