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In the present study, we compared the enzyme kinetics and allosteric modulation of six recombinant AMPK isoforms, \u03b11\u03b21\u03b31, \u03b11\u03b22\u03b31, \u03b11\u03b22\u03b33, \u03b12\u03b21\u03b31, \u03b12\u03b22\u03b31 and \u03b12\u03b22\u03b33 using known activators, A769662 and AMP. The \u03b11-containing complexes exhibited higher specific activities and lower Km values for a widely used peptide substrate (SAMS) compared with \u03b12-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 \u03b12-complexes were \u223c25-fold more sensitive than \u03b11-complexes to dephosphorylation of a critical threonine on their activation loop (pThr172\/174). However, \u03b12-complexes were more readily activated by AMP than \u03b11-complexes. Compared with \u03b21-containing heterotrimers, \u03b22-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.<\/jats:p>","DOI":"10.1042\/bj20151051","type":"journal-article","created":{"date-parts":[[2015,12,4]],"date-time":"2015-12-04T02:58:21Z","timestamp":1449197901000},"page":"581-592","update-policy":"https:\/\/doi.org\/10.1042\/crossmark_policy","source":"Crossref","is-referenced-by-count":66,"title":["Probing the enzyme kinetics, allosteric modulation and activation of \u03b11- and \u03b12-subunit-containing AMP-activated protein kinase (AMPK) heterotrimeric complexes by pharmacological and physiological activators"],"prefix":"10.1042","volume":"473","author":[{"given":"Francis","family":"Rajamohan","sequence":"first","affiliation":[{"name":"Department of World Wide Medicinal Chemistry, Pfizer Global Research and Development, Eastern Point Road, Groton, CT 06340, U.S.A."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Allan\u00a0R.","family":"Reyes","sequence":"additional","affiliation":[{"name":"CVMED Research unit, Pfizer Global Research and Development, 620 Memorial Drive, Cambridge, MA 02139, U.S.A."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Richard\u00a0K.","family":"Frisbie","sequence":"additional","affiliation":[{"name":"The Department of Pharmacokinetics Dynamics and Metabolism, Pfizer Global Research and Development, Eastern Point Road, Groton, CT 06340, U.S.A."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lise\u00a0R.","family":"Hoth","sequence":"additional","affiliation":[{"name":"Department of World Wide Medicinal Chemistry, Pfizer Global Research and Development, Eastern Point Road, Groton, CT 06340, U.S.A."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Parag","family":"Sahasrabudhe","sequence":"additional","affiliation":[{"name":"Department of World Wide Medicinal Chemistry, Pfizer Global Research and Development, Eastern Point Road, Groton, CT 06340, U.S.A."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rachelle","family":"Magyar","sequence":"additional","affiliation":[{"name":"Department of World Wide Medicinal Chemistry, Pfizer Global Research and Development, Eastern Point Road, Groton, CT 06340, U.S.A."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"James\u00a0A.","family":"Landro","sequence":"additional","affiliation":[{"name":"CVMED Research unit, Pfizer Global Research and Development, 620 Memorial Drive, Cambridge, MA 02139, U.S.A."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jane\u00a0M.","family":"Withka","sequence":"additional","affiliation":[{"name":"Department of World Wide Medicinal Chemistry, Pfizer Global Research and Development, Eastern Point Road, Groton, CT 06340, U.S.A."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nicole\u00a0L.","family":"Caspers","sequence":"additional","affiliation":[{"name":"Department of World Wide Medicinal Chemistry, Pfizer Global Research and Development, Eastern Point Road, Groton, CT 06340, U.S.A."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Matthew\u00a0F.","family":"Calabrese","sequence":"additional","affiliation":[{"name":"Department of World Wide Medicinal Chemistry, Pfizer Global Research and Development, Eastern Point Road, Groton, CT 06340, U.S.A."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jessica","family":"Ward","sequence":"additional","affiliation":[{"name":"CVMED Research unit, Pfizer Global Research and Development, 620 Memorial Drive, Cambridge, MA 02139, U.S.A."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ravi\u00a0G.","family":"Kurumbail","sequence":"additional","affiliation":[{"name":"Department of World Wide Medicinal Chemistry, Pfizer Global Research and Development, Eastern Point Road, Groton, CT 06340, U.S.A."}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"288","published-online":{"date-parts":[[2016,2,24]]},"reference":[{"key":"2021112216344756600_B1","doi-asserted-by":"publisher","first-page":"821","DOI":"10.1146\/annurev.biochem.67.1.821","article-title":"The AMP-activated\/SNF1 protein kinase subfamily: metabolic sensors of the eukaryotic cell?","volume":"67","author":"Hardie","year":"1998","journal-title":"Ann. 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