AN INVITRO NOVEL MECHANISM OF REGULATING THE ACTIVITY OF PYRUVATE KINASE-M2 BY THYROID-HORMONE AND FRUCTOSE-1,6-BISPHOSPHATE

We have recently shown that the cytosolic thyroid hormone binding protein (p58-M2) in human epidermoid carcinoma A431 cells is a monomer of pyruvate kinase, subtype M2 (PKM2). To characterize further the molecular properties of p58-M2, we overexpressed p58-M2 in Escherichia coli and purified it to homogeneity. At 22-degrees-C, the monomeric p58-M2 exhibited kinase activity with an apparent V(max) of 22 +/- 9 units/mg. The Km for adenosine diphosphate (ADP) and phosphoenolpyruvate (PEP) are 3.85 +/- 2.4 and 1.55 +/- 0.73 mM, respectively. Upon activation by fructose 1,6-bisphosphate (Fru-1,6-P2), V(max) and K(m) for ADP and PEP were changed to 490 +/- 27 units/mg and 0.63 +/- 0.09 and 0.1 3 +/- 0.01 mM, respectively. These results indicated that p58-M2 has intrinsic kinase activity. Analysis of the molecular size indicated that the activation of p58-M2 by Fru-1,6-P2 resulted in the association of the monomeric p58-M2 to the tetrameric PKM2. p58-M2 bound to 3,3',5-triiodo-L-thyronine (T3) (K(a) = 1.7 X 10(7) M-1) and exhibited analogue specificity, whereas PKM2 did not bind thyroid hormone. The order of binding affinity was L-T3 > L-thyroxine > 3,3',5-triiodothyropropionic acid > 3'-isopropyl-3,5-triiodo-L-thyronine > 3',5',3-triiodo-L-thyronine. Binding of T3 and its analogues resulted in the inhibition of the kinase activity of p58-M2. The order of kinase inhibitory activity and preventing its association to tetrameric PKM2 was parallel to that of binding activity. The present study demonstrated that, in vitro, the molecular mechanism by which Fru-1,6-p2 induced activation of PK activity is by tetramer formation. Furthermore, thyroid hormone plays a role in regulating the enzymatic activity of PKM2.