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首页> 外文期刊>Biochemistry >A pre-steady state analysis of ligand binding to human glucokinase: evidence for a preexisting equilibrium.
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A pre-steady state analysis of ligand binding to human glucokinase: evidence for a preexisting equilibrium.

机译：配体与人葡萄糖激酶结合的稳态前分析：已有平衡的证据。

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Cooperativity with glucose is a key feature of human glucokinase (GK), allowing its crucial role as a glucose sensor in hepatic and pancreatic cells. We studied the changes in enzyme intrinsic tryptophan fluorescence induced by binding of different ligands to this monomeric enzyme using stopped-flow and equilibrium binding methods. Glucose binding data under pre-steady state conditions suggest that the free enzyme in solution is in a preexisting equilibrium between at least two conformers (super-open and open) which differ in their affinity for glucose (Kd* = 0.17 +/- 0.02 mM and Kd = 73 +/- 18 mM). Increasing the glucose concentration changes the ratio of the two conformers, thus yielding an apparent Kd of 3 mM (different from a Km of 7-10 mM). The rates of conformational transitions of free and GK complexed with sugar are slow and during catalysis are most likely affected by ATP binding, phosphate transfer, and product release steps to allow the kcat to be 60 s-1. The ATP analogue PNP-AMP binds to free GK (super-open) and GK-glucose (open) complexes with comparable affinities (Kd = 0.23 +/- 0.02 and 0.19 +/- 0.08 mM, respectively). However, cooperativity with PNP-AMP observed under equilibrium binding conditions in the presence of glucose (Hill slope of 1.6) is indicative of further complex tightening to the closed conformation. Another physiological modulator (inhibitor), palmitoyl-CoA, binds to GK with similar characteristics, suggesting that conformational changes induced upon ligand binding are not restricted by an active site ligand. In conclusion, our data support control of GK activity and Km through the ratio of distinct conformers (super-open, open, and closed) through either substrate or other ligand binding and/or dissociation.
机译：与葡萄糖的协同作用是人葡萄糖激酶（GK）的关键特征，使其在肝细胞和胰腺细胞中作为葡萄糖传感器发挥关键作用。我们使用停止流和平衡结合方法研究了不同配体与该单体酶结合引起的酶固有色氨酸荧光的变化。在稳态之前的葡萄糖结合数据表明，溶液中的游离酶处于至少两个构象体（超开放和开放）之间的既有平衡状态，这两个构象对葡萄糖的亲和力不同（Kd * = 0.17 +/- 0.02 mM和Kd = 73 +/- 18 mM）。葡萄糖浓度的增加会改变两个构象异构体的比例，从而产生3 mM的表观Kd（与7-10 mM的Km不同）。游离和GK与糖复合的构象转变速率很慢，并且在催化过程中最有可能受到ATP结合，磷酸盐转移和产物释放步骤的影响，以使kcat为60 s-1。 ATP类似物PNP-AMP以可比的亲和力（分别为Kd = 0.23 +/- 0.02和0.19 +/- 0.08 mM）与游离GK（超开放）和GK-葡萄糖（开放）复合物结合。然而，在葡萄糖存在的情况下，在平衡结合条件下观察到的与PNP-AMP的协同作用（希尔斜率为1.6）表明进一步复杂的收紧至闭合构象。另一种生理调节剂（抑制剂），棕榈酰辅酶A，以相似的特性与GK结合，表明配体结合后诱导的构象变化不受活性位点配体的限制。总之，我们的数据支持通过底物或其他配体结合和/或解离的不同构象体（超开放，开放和封闭）的比率控制GK活性和Km。

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