Figure 2. Time-dependence of PDC inactivation by PDK1 and PDK2.

(Top) PDK1 (3.0 μg, 0.1 μM) in 50 mM KH₂PO₄ (pH 7.5) supplemented with 0.5 mM ThDP, 1.0 mM MgCl₂, 4.0 mM DTT and 0.1 mM EDTA was incubated with E1 (75 μg, 1.95 μM) in the presence of either L1L2S (3.0 μg, 0.3 μM) (line 6, ); E2⋅E3BP (3.0 μg, 0.2 μM), (line 5, ); L2S (2.4 μg, 0.5 μM), (line 4, ◽); L1 (3.0 μg, 1.0 μM) (line 3, ); L3S’ (1.0 μg, 0.5 μM) (line 2, ) or with no lipoyl domain source (line 1, ●). Phosphorylation was initiated by ATP (0.5 mM) at 23 °C. (Bottom) PDK2 (6.6 μg, 0.48 μM) in 50 mM KH₂PO₄ (pH 7.5) supplemented with 0.5 mM ThDP, 1.0 mM MgCl₂, 4.0 mM DTT, and 0.1 mM EDTA was incubated with E1 (80 μg, 3.5 μM) with no lipoyl domain source (line 6, ●); in the presence of either: L3S’ (1.0 μg, 0.24 μM) (line 5, ); E2· E3BP (2.0 μg, 0.22 μM) (line 4, ); L1L2S (1.2 μg, 0.22 μM), (line 3, ); L2S (0.8 μg, 0.22 μM) (line 2, ◽) and L1 (0.4 μg, 0.22 μM), (line 1, ). Phosphorylation was initiated by ATP (2.0 mM) at 30 °C. Aliquots (1 μg E1) were withdrawn at different times and were mixed with E2⋅E3BP and E3 at a mass ratio of E1:E2·E3BP: E3 of 1:3:3 to measure PDC activity.