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 thalianaα2β2-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 E1α, is phosphorylated. In addition to Ser292, Asp295 and Gly297 are highly conserved in the E1α 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 E1α. 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.

“Scanning mutagenesis” of the amino acid sequences flanking phosphorylation site 1 of the mitochondrial pyruvate dehydrogenase complex

“…Previous studies of the D295N change using peptide substrates with the mammalian PDK showed substantially reduced phosphorylation of S 292 , and it was proposed that Asp 295 is involved with PDK binding (Mullinax et al, 1985 ). The results of Mullinax et al ( 1985 ) were recently verified by Hirani et al ( 2011 ) using recombinant A. thaliana PDC-E1 and PDK. The D295N, D295A, and D295L site-directed mutants all had greatly reduced phosphorylation of E1α.…”

“…During the genome-sequencing era it has been established that the position of Ser 292 and the sequences of the flanking amino acids are highly conserved in mtPDC E1α sequences. When directly studied, it has been verified that Ser 292 was phosphorylated by the intrinsic PDK (e.g., Bykova et al, 2003 ; Hirani et al, 2011 ).…”

“…Hirani et al (2011) reported that with the G297S and G297D site-directed mutants the phosphorylation of Ser 292 was only inhibited approximately 30%. The phylogenetic sequence alignments revealed an absolute conservation of Gly at position 297 (Figure 1).…”

“…Our “scanning mutagenesis” strategy, using synthetic peptides, recombinant PDK, and the quantitative KiC assay, yielded a set of results that would have been at least difficult to obtain using site-directed mutagenesis of the intact protein sequence because of the occurrence of competing effects upon catalytic activity (e.g., Hirani et al, 2011 ). As suggested by the results from comparative sequence analyses, the site 1 flanking sequences tolerate little change.…”

“…There have been a very limited number of studies aimed at characterizing the effects of changes to amino acids flanking site 1 (Sugden et al, 1979 ; Mullinax et al, 1985 ; Hirani et al, 2011 ). Herein we report results from analysis of a series of systematic changes to Ser-flanking residues on phosphorylation of site 1 by PDK, using the synthetic peptide-based kinase client (KiC) assay (Huang et al, 2010 ).…”