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 homoeostasis. AMPK exists as a heterotrimeric complex, composed of a catalytic subunit (alpha) and two regulatory subunits (beta and gamma), each present as multiple isoforms. In the present study, we compared the enzyme kinetics and allosteric modulation of six recombinant AMPK isoforms, alpha 1 beta 1 gamma 1, alpha 1 beta 2 gamma 1, alpha 1 beta 2 gamma 3, alpha 2 beta 1 gamma 1, alpha 2 beta 2 gamma 1 and alpha 2 beta 2 gamma 3 using known activators, A769662 and AMP. The alpha 1-containing complexes exhibited higher specific activities and lower K-m values for a widely used peptide substrate (SAMS) compared with alpha 2-complexes. Surface plasmon resonance (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 alpha 2-complexes were similar to 25-fold more sensitive than alpha 1-complexes to dephosphorylation of a critical threonine on their activation loop (pThr(172/174)). However, alpha 2-complexes were more readily activated by AMP than alpha 1-complexes. Compared with beta 1-containing heterotrimers, beta 2=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.