Asp295 Stabilizes the Active-Site Loop Structure of Pyruvate Dehydrogenase, Facilitating Phosphorylation of Ser292 by Pyruvate Dehydrogenase-Kinase
Figure 5
Phosphorylation of AtPDC E1α active-site loop mutants by AtPDK. (a) (i) Autoradiograph comparing phosphorylation of WT AtPDC E1 and the Asp295 mutant. (a) (ii) Autoradiograph comparing phosphorylation of WT AtPDC E1 and the Gly297 mutant. One μg each of the WT and mutant proteins was incubated with 0.25 μg AtPDK and 200 μM [γ-32P]ATP at 30°C for 90 min. In each case the entire reaction mixture was loaded onto the gels. After electrophoresis, all gels were exposed to film for 3 h. (b) Relative (to WT) phosphorylation of the Asp295 and Gly297 mutants is indicated. Data are mean values from at least three separate determinations ± SEM. The value for 100% of WT phosphorylation was μmol 32P μmol AtPDC E1−1. (c) Time-course of phosphorylation. The WT AtPDC E1 (▴), and the G297D (□), D295A (○), and D295N (♦) mutant proteins, were phosphorylated as described in (a). Reactions volumes were 100 μL. Samples of 20 μL were removed at each time point, mixed with SDS-sample buffer, heated, and then resolved by SDS-PAGE.
