Stimulation of bovine heart pyruvate dehydrogenase kinase by α-ketoisovalerate in the presence of thiamin pyrophosphate

Purified bovine heart pyruvate dehydrogenase complex was used to study pyruvate dehydrogenase kinase (PDH kinase) regulation. Previously, they showed that KCl and NH4Cl stimulate PDH kinase, and that thiamin pyrophosphate over the range of 1-80 μM completely blocks PDH kinase stimulation by 20 mM KCl, whereas thiamin pyrophosphate has very little inhibitory effect in the absence of KCl. Under inhibitory conditions, 100 μM thiamin pyrophosphate and 20 mM KCl, addition of 1 mM α-ketoisovalerate stimulated PDH kinase activity 1.5-fold. Addition of 1 mM α-ketoisovalerate had a similar stimulatory effect in the presence of 1 mM NH4Cl and 100 μM thiamin pyrophosphate. Half-maximal stimulation occurred at about 200 μM α-ketoisovalerate in both cases. Inhibition by thiamin pyrophosphate was uncompetitive, and the effect of α-ketoisovalerate on thiamin pyrophosphate inhibition was to shift both Vm and Km toward control values without changing the slope of the reciprocal plot. After incubation of pyruvate dehydrogenase complex with α-ketoisovalerate and gel filtration on Sephadex G-25 to remove α-ketoisovalerate, PDH kinase activity remained stimulated relative to controls incubated with only buffer. In bovine heart mitochondria solubilized in 0.2% Triton X-100 and incubated with [γ-32P]ATP, α-ketoisovalerate stimulated PDH phosphorylation in the presence of thiamin pyrophosphate, as detected by SDS-PAGE and autoradiography. The results indicate that acylation of pyruvate dehydrogenase complex results in PDH kinase stimulation, but only in the presence of monovalent cation and thiamin pyrophosphate. Furthermore, the results also suggest that this effect may be relevant under more in vivo conditions