AN INVITRO NOVEL MECHANISM OF REGULATING THE ACTIVITY OF PYRUVATE KINASE-M2 BY THYROID-HORMONE AND FRUCTOSE-1,6-BISPHOSPHATE
AN INVITRO NOVEL MECHANISM OF REGULATING THE ACTIVITY OF PYRUVATE KINASE-M2 BY THYROID-HORMONE AND FRUCTOSE-1,6-BISPHOSPHATE
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DOI：
10.1021/bi00243a010
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发表时间：
1991-07-23
期刊：
影响因子：
2.9
通讯作者：
CHENG, SY
中科院分区：
文献类型：
--
作者：
ASHIZAWA, K;MCPHIE, P;CHENG, SY
我们最近发现，在人表皮样癌A431细胞中的胞浆甲状腺激素结合蛋白（p58-M2）是丙酮酸激酶M2亚型（PKM 2）的单体。为了进一步表征p58-M2的分子特性，我们在大肠杆菌中过表达p58-M2并将其纯化至均一。在22 ℃时，单体p58-M2表现出激酶活性，表观V（max）为22 +/- 9单位/mg。腺苷二磷酸（ADP）和磷酸烯醇丙酮酸（PEP）的Km分别为3.85 +/- 2.4和1.55 +/- 0.73 mM。在被果糖1，6-二磷酸（Fru-1，6-P2）激活后，ADP和PEP的V（max）和K（m）分别变为490 +/- 27单位/mg和0.63 +/- 0.09和0.13 +/- 0.01 mM。这些结果表明p58-M2具有内在激酶活性。分子大小的分析表明，通过Fru-1，6-P2激活p58-M2导致单体p58-M2与四聚体PKM 2的缔合。 p58-M2与3，3 '，5-三碘-L-甲状腺原氨酸（T3）结合（K（a）= 1.7 × 10（7）M-1）并显示类似物特异性，而PKM 2不与甲状腺激素结合。结合亲和力大小顺序为：L-T3 > L-甲状腺素> 3，3 '，5-三碘甲状腺丙酸> 3'-异丙基-3，5-三碘-L-甲状腺原氨酸> 3 '，5'，3-三碘-L-甲状腺原氨酸。T3及其类似物的结合导致p58-M2激酶活性的抑制。激酶抑制活性和防止其与四聚体PKM 2结合的顺序与结合活性的顺序平行。本研究表明，在体外，Fru-1，6-p2诱导PK活性激活的分子机制是通过四聚体形成。此外，甲状腺激素在调节PKM 2的酶活性中起作用。
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.