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/threonine protein kinase that serves as a pleotropic regulator of whole body energy homeostasis. AMPK exists 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 kinetics and allosteric modulation of six recombinant AMPK isoforms, 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 threonine on 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 AMPK isoforms are less sensitive to activation by A769662, a synthetic activator. These data demonstrate that ligand induced activation of AMPK isoforms may vary significantly based on their AMPK subunit composition. Our studies provide insights for the design of isoform-selective AMPK activators for the treatment of metabolic diseases.