Marked differences between two isoforms of human pyruvate dehydrogenase kinase

Area of Science:

• Biochemistry
• Enzymology
• Metabolic Regulation

Background:

• Pyruvate dehydrogenase kinase (PDK) regulates pyruvate dehydrogenase (E1) activity, a key enzyme in cellular respiration.
• PDK isoforms 2 and 3 interact with dihydrolipoyl acetyltransferase (E2) components, influencing metabolic flux.
• Understanding these interactions is crucial for comprehending cellular energy homeostasis and metabolic control.

Purpose of the Study:

• To investigate the differential activation mechanisms of PDK isoforms 2 and 3 by dihydrolipoyl acetyltransferase (E2) components.
• To elucidate how binding to E2 lipoyl domains affects the phosphorylation rates of the pyruvate dehydrogenase (E1) component.
• To characterize the regulatory responses of PDK2 and PDK3 to effectors like pyruvate, dichloroacetate (DCA), and acetylated lipoyl domains.

Main Methods:

• Co-expression of human PDK isoforms 2 and 3 with chaperonins GroEL/GroES for purification.
• Affinity tag-free purification of recombinant PDK isoforms.
• Enzyme kinetics assays to determine activation and inhibition parameters by E2 components and effectors.

Main Results:

• PDK3 demonstrated significant activation by the free L2 lipoyl domain of E2, with altered kinetics for E1 phosphorylation.
• PDK3, once activated, showed reduced sensitivity to pyruvate and dichloroacetate (DCA).
• PDK2 activation was dependent on the assembled E2 60-mer, leading to enhanced sensitivity to pyruvate and DCA inhibition.

Conclusions:

• PDK3 exhibits direct activation by the L2 domain, displaying relative insensitivity to feed-forward and feed-back regulators.
• PDK2 activation requires the complete E2 assembly, rendering it highly responsive to regulatory effectors.
• These findings highlight distinct regulatory strategies employed by PDK2 and PDK3 in controlling pyruvate dehydrogenase complex activity.