Probing the enzyme kinetics, allosteric modulation and activation of α1- and α2-subunit-containing AMP-activated protein kinase (AMPK) heterotrimeric complexes by pharmacological and physiological activators

AMP-activated protein kinase( AMPK) is a serine/ threonineprotein kinase that serves as a pleotropic regulator of whole body energy homeostasis. AMPKexists as a heterotrimeric complex, composed of a catalytic subunit (a) and two regulatory subunits (b and g), each present as multiple isoforms. In this study, we compared the enzyme kineticsand allosteric modulationof six recombinant AMPKisoforms, a1b1g1, a1b2g1, a1b2g3, a2b1g1, a2b2g1, and a2b2g3 using known activators, A769662 and AMP. The a1-containing complexes exhibited higher specific activities and lower K m values for a widely used peptide substrate (SAMS) compared to a2-complexes. SPR-based direct binding measurements revealed biphasic binding modes with two distinct equilibrium binding constants for AMP, ADP and ATP across all isoforms tested. The a2-complexes were ~25-fold more sensitive than a1-complexes todephosphorylation of a critical threonineon their activation loop (pT 172/174 ). However, a2-complexes were more readily activated by AMP than a1-complexes. Compared to β1-containing heterotrimers, β2-containing AMPKisoforms are less sensitive to activation by A769662, a synthetic activator. These data demonstrate that ligand induced activation of AMPKisoforms may vary significantly based on their AMPKsubunit composition. Our studies provide insights for the design of isoform-selective AMPKactivators for the treatment of metabolic diseases.