Department of Biochemistry and Biophysics and Diabetes Research Center, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
Biochem J. 2011 Dec 1;440(2):203-15. doi: 10.1042/BJ20110440.
GK (glucokinase) is activated by glucose binding to its substrate site, is inhibited by GKRP (GK regulatory protein) and stimulated by GKAs (GK activator drugs). To explore further the mechanisms of these processes we studied pure recombinant human GK (normal enzyme and a selection of 31 mutants) using steady-state kinetics of the enzyme and TF (tryptophan fluorescence). TF studies of the normal binary GK-glucose complex corroborate recent crystallography studies showing that it exists in a closed conformation greatly different from the open conformation of the ligand-free structure, but indistinguishable from the ternary GK-glucose-GKA complex. GKAs did activate and GKRP did inhibit normal GK, whereas its TF was doubled by glucose saturation. However, the enzyme kinetics, GKRP inhibition, TF enhancement by glucose and responsiveness to GKA of the selected mutants varied greatly. Two predominant response patterns were identified accounting for nearly all mutants: (i) GK mutants with a normal or close to normal response to GKA, normally low basal TF (indicating an open conformation), some variability of kinetic parameters (k(cat), glucose S(0.5), h and ATP K(m)), but usually strong GKRP inhibition (13/31); and (ii) GK mutants that are refractory to GKAs, exhibit relatively high basal TF (indicating structural compaction and partial closure), usually show strongly enhanced catalytic activity primarily due to lowering of the glucose S(0.5), but with reduced or no GKRP inhibition in most cases (14/31). These results and those of previous studies are best explained by envisioning a common allosteric regulator region with spatially non-overlapping GKRP- and GKA-binding sites.
GK（葡萄糖激酶）通过葡萄糖与其底物结合位点的结合而被激活，被 GKRP（葡萄糖激酶调节蛋白）抑制，被 GKAs（葡萄糖激酶激活剂药物）刺激。为了进一步探索这些过程的机制，我们使用酶的稳态动力学和 TF（色氨酸荧光）研究了纯重组人 GK（正常酶和 31 种突变体的选择）。TF 研究正常二元 GK-葡萄糖复合物证实了最近的晶体学研究，表明它存在于一种与无配体结构的开放构象大不相同的封闭构象，但与三元 GK-葡萄糖-GKA 复合物无法区分。GKAs 确实激活了正常 GK，而 GKRP 则抑制了正常 GK，而葡萄糖则使 TF 增加了一倍。然而，所选突变体的酶动力学、GKRP 抑制、葡萄糖对 TF 的增强以及对 GKA 的反应性差异很大。确定了两种主要的反应模式，几乎可以解释所有的突变体：（i）对 GKA 具有正常或接近正常反应的 GK 突变体，正常的 TF 基础值（表示开放构象）较低，动力学参数（k(cat)、葡萄糖 S(0.5)、h 和 ATP K(m)）的某些可变性，但通常具有强烈的 GKRP 抑制（13/31）；和（ii）对 GKA 无反应的 GK 突变体，表现出相对较高的基础 TF（表示结构紧凑和部分关闭），通常表现出强烈增强的催化活性，主要是由于降低了葡萄糖 S(0.5)，但在大多数情况下降低了 GKRP 抑制（14/31）。这些结果和以前的研究结果最好通过设想一个具有空间上不重叠的 GKRP 和 GKA 结合位点的共同变构调节剂区域来解释。
