Oxford Centre for Diabetes Endocrinology and Metabolism, University of Oxford, Oxford OX3 7LJ, United Kingdom.
J Biol Chem. 2011 May 27;286(21):19118-26. doi: 10.1074/jbc.M111.223362. Epub 2011 Mar 29.
Type 2 diabetes is a global problem, and current ineffective therapeutic strategies pave the way for novel treatments like small molecular activators targeting glucokinase (GCK). GCK activity is fundamental to beta cell and hepatocyte glucose metabolism, and heterozygous activating and inactivating GCK mutations cause hyperinsulinemic hypoglycemia (HH) and maturity onset diabetes of the young (MODY) respectively. Over 600 naturally occurring inactivating mutations have been reported, whereas only 13 activating mutations are documented to date. We report two novel GCK HH mutations (V389L and T103S) at residues where MODY mutations also occur (V389D and T103I). Using recombinant proteins with in vitro assays, we demonstrated that both HH mutants had a greater relative activity index than wild type (6.0 for V389L, 8.4 for T103S, and 1.0 for wild type). This was driven by an increased affinity for glucose (S(0.5), 3.3 ± 0.1 and 3.5 ± 0.1 mm, respectively) versus wild type (7.5 ± 0.1 mm). Correspondingly, the V389D and T103I MODY mutants had markedly reduced relative activity indexes (<0.1). T103I had an altered affinity for glucose (S(0.5), 24.9 ± 0.6 mm), whereas V389D also exhibited a reduced affinity for ATP and decreased catalysis rate (S(0.5), 78.6 ± 4.5 mm; ATP(K(m)), 1.5 ± 0.1 mm; K(cat), 10.3 ± 1.1s(-1)) compared with wild type (ATP(K(m)), 0.4 ± <0.1; K(cat), 62.9 ± 1.2). Both Thr-103 mutants showed reduced inhibition by the endogenous hepatic inhibitor glucokinase regulatory protein. Molecular modeling demonstrated that Thr-103 maps to the allosteric activator site, whereas Val-389 is located remotely to this position and all other previously reported activating mutations, highlighting α-helix 11 as a novel region regulating GCK activity. Our data suggest that pharmacological manipulation of GCK activity at locations distal from the allosteric activator site is possible.
2 型糖尿病是一个全球性问题，目前无效的治疗策略为靶向葡萄糖激酶 (GCK) 的小分子激活剂等新疗法铺平了道路。GCK 活性对β细胞和肝细胞的葡萄糖代谢至关重要，杂合激活和失活 GCK 突变分别导致高胰岛素血症性低血糖 (HH) 和青少年发病的成年型糖尿病 (MODY)。目前已报道了超过 600 种天然失活突变，而迄今为止仅记录了 13 种激活突变。我们报道了两种新的 GCK HH 突变 (V389L 和 T103S)，它们位于 MODY 突变发生的残基处 (V389D 和 T103I)。使用体外测定的重组蛋白，我们证明这两种 HH 突变体的相对活性指数都高于野生型 (V389L 为 6.0，T103S 为 8.4，野生型为 1.0)。这是由于对葡萄糖的亲和力增加所致 (S(0.5)，分别为 3.3 ± 0.1 和 3.5 ± 0.1 mm，而野生型为 7.5 ± 0.1 mm)。相应地，V389D 和 T103I MODY 突变体的相对活性指数显著降低 (<0.1)。T103I 对葡萄糖的亲和力发生改变 (S(0.5)，24.9 ± 0.6 mm)，而 V389D 也表现出对 ATP 的亲和力降低和催化速率降低 (S(0.5)，78.6 ± 4.5 mm；ATP(K(m))，1.5 ± 0.1 mm；K(cat)，10.3 ± 1.1 s(-1))，与野生型相比 (ATP(K(m))，0.4 ± <0.1；K(cat)，62.9 ± 1.2)。两种 Thr-103 突变体对内源性肝抑制剂葡萄糖激酶调节蛋白的抑制作用均降低。分子建模表明 Thr-103 位于变构激活剂位点，而 Val-389 位于该位置之外，并且所有其他先前报道的激活突变都位于此，突出了 α-螺旋 11 是调节 GCK 活性的新区域。我们的数据表明，在变构激活剂位点以外的位置对 GCK 活性进行药理学操作是可能的。
