Asp295 Stabilizes the Active-Site Loop Structure of Pyruvate Dehydrogenase, Facilitating Phosphorylation of Ser292 by Pyruvate Dehydrogenase-Kinase

Figure 4

Analysis of the AtPDC E1α S298A mutant phosphorylation site by mass spectrometry. (a) MALDI-TOF MS analysis of tryptic peptides from the AtPDC E1α S298A mutant. The 1656.9 m/z ion corresponds to the phosphopeptide Tyr286 to Arg301. (b) Fragmentation of the m/z 1656.9 peptide by nanoelectrospray tandem MS. Fragment ion spectrum from the triple-charge molecular ion at m/z 553.55. The ions b5⁺ (662.25), b6⁺ (793.23), and y10⁺ (1164.41) indicate P-Ser292. The asterisks mark the fragment ions from the S298A-methylated peptide that was also present in the parent ion envelope at (M + 3H)³⁺ of 553.95 Da. (c) Expanded spectrum of the y10⁺ ion.