Asp295 Stabilizes the Active-Site Loop Structure of Pyruvate Dehydrogenase, Facilitating Phosphorylation of Ser292 by Pyruvate Dehydrogenase-Kinase
Figure 3
Phosphorylation of recombinant AtPDC E1 by recombinant AtPDK. (a) Autoradiograph of the in vitro phosphorylation of wild-type (WT) AtE1α, and the S292A and S298A mutants, by recombinant AtPDK. One μg of E1 was incubated with 0.25 μg PDK and 200 μM [γ-32P]ATP, at 30°C for 90 min. When the film was developed after 3 h, phosphorylation of the WT and the S298A proteins was visible, while no phosphorylation was observed for the S292A and the double mutants. Furthermore, no phosphorylation of the S292A or double mutants was observed when the same film was developed after 16 h. (b) Phosphorylation of the mutant proteins relative to WT. The 100% phosphorylation value of WT was μmol 32P mol AtPDC E1−1. Data are the mean values from at least three separate determinations ± SEM.
