| The expression vector of human pyruvate dehydrogenase kinase isoform 4 (PDK4) was reengineered to overcome the proteolytic degradation of PDK4 in E. coli. PDK4 was successfully expressed and purified to near homogeneity. The production of soluble and active PDK4 was facilitated with the coexpression of GroELS chaperonins.;At high E1 levels from 1.5 to 7 muM, PDK4 gave high specific activities with free pyruvate dehydrogenase (E1) and addition of dihydrolipoyl transacetylase (E2) 60mer did not enhance the activity in this E1 range. However, when the concentration of E1 was lowered to less than 1 muM, E2-E3BP activated PDK4 activity by 2--3 fold through the specific interactions between PDK4 and outer lipoyl domain of E2 or lipoyl domain of dihydrolipoyl reductase binding protein (E3BP). Activation of PDK4 by an E2 mutant lacking the E1 binding domain (DeltaBE2) in the presence of E1 was much weaker than that by E2-E3BP-E1 complex indicating that E1 also contributed to the interaction between PDK4 and E1-E2-E3BP complex. Using analytical ultracentrifugation, we found that the interactions between PDK4 and E1-E2-E3BP complex were weak. Besides enhancing the E1-phosphorylation rate, E2 also increased the extent of E1-phosphorylation by PDK4.;PDK4 activity was inhibited strongly by Cl- and moderately by K+. Elevated salt, especially 100 mM K +, caused a pronounced lowering of the concentration of E1 or E2-bound E1 giving the half maximum activity. Potassium also significantly decreased the Km of PDK4 for ATP and decreased the Ki of PDK4 for inhibitors such as ADP, pyruvate and dichloroacetic acid (DCA).;DCA, an analog of pyruvate, was a potent inhibitor of PDK4 while the native inhibitor, pyruvate, only moderately inhibited PDK4 activity. Chloride ion hindered DCA inhibition and even more effectively, pyruvate inhibition. The combination of DCA and ADP inhibited PDK4 synergistically, while combination of pyruvate and ADP only gave additive inhibition.;Under a variety of buffer and effector conditions, PDK4 was not strongly stimulated by NADH, acetyl-CoA and their combination. This indicated that the rate of inactivation of PDC by PDK4 was insensitive to increased fatty acid oxidation. |
