Ligand Binding and Kinetic Studies on Human Erythrocyte Glucose-6-Phosphate Dehydrogenase Dimers

该项目的目标是使用一种新的方法来简化 人红细胞葡萄糖-6-动力学和热力学研究 磷酸脱氢酶（G6 PD），涉及控制 酶的结合状态 G6 PD是一种关键酶， 己糖磷酸途径和超过370种遗传变异的 人红细胞酶已被鉴定。人士 G6 PD缺乏症可在暴露于 各种药物或食物。 大量的基因变异， 许多具有降低的活性，使得G6 PD成为一个有吸引力的靶点， 克隆和测序。 对变化的解释 氨基酸序列依赖于G6 PD的测量 生化特性 不幸的是，缺乏 协议的许多基本酶的性质， 红细胞G6 PD，即底物和红细胞G6 PD的结合。 细胞间分子对酶动力学的影响。 在 与先前对酵母G6 PD的工作类似，该提议的假设 过去对人红细胞的动力学和热力学研究 G6 PD测量了G6 PD二聚体和 四聚体，其中的每一个可以具有不同的生物化学性质， 特性. 关于人类红细胞缺乏一致意见， G6 PD的生物化学特性会出现，因为相对的 G6 PD二聚体和四聚体的量在动力学过程中发生变化 或热力学测量。 因此 建议将侧重于测量的生化特性， G6 PD二聚体，假定存在于 红细胞 RUI提案的三个具体目标是：（1） 来测量烟酰胺腺嘌呤二核苷酸的数量 磷酸（NADP）和葡萄糖-6-磷酸（G6 P）分子结合 与G6 PD的结合常数以及这些结合常数的大小 两个分子。该项目将直接测量 放射性标记的G6 P或NADP的量，结合到 酵素 该提案还将（2）定量研究 ATP和2，3抑制红细胞G6 PD动力学 二磷酸甘油酸，两个提出生理上重要的 G6 PD抑制剂。 第三（3）项具体目标将衡量 二聚体-四聚体缔合常数，使用大区域（平台） 尺寸排阻柱上的HPLC实验。

The project's goal is to use a new approach to simplify the kinetic and thermodynamic study of human erythrocyte glucose-6- phosphate dehydrogenase (G6PD) that involves the control of the enzyme's state of association. G6PD is a key enzyme in the hexosemonophosphate pathway and over 370 genetic variants of the human erythrocyte enzyme have been identified. Individuals with G6PD deficiencies can suffer hemolytic crisis on exposure to a variety of drugs or foods. The large number of genetic variants, many with reduced activity, mae G6PD an attractive target for cloning and sequencing. The interpretation of the changes in amino acid sequence are dependent on the measurement of G6PD's biochemical properties. Unfortunately, there is a lack of agreement on many of the basic enzymatic properties of erythrocyte G6PD, namely, the binding of substrates and the effect of intercellular molecules on the enzyme's kinetics. In analogy with prior work on yeast G6PD, this proposal's hypothesis is past kinetic and thermodynamic studies of human erythrocyte G6PD measured the properties of a mixture of G6PD dimers and tetramers, each of which may have different biochemical properties. This lack of agreement concerning human erythrocyte G6PD's biochemical properties would arise because the relative amounts of G6PD dimers and tetramers changed during the kinetic or thermodynamic measurements. Thus proposal will focus upon measuring the biochemical properties of the G6PD dimer, the form presumed to be present in the erythrocyte. The three specific aims of the RUI proposal are (1) to measure the number of nicotinamide adenine dinucleotide phosphate (NADP) and glucose-6-phosphate (G6P) molecules bound to G6PD and the magnitude of the association constant for these two molecules. The project will directly measure the equilibrium quantity of radioactively labeled G6P, or NADP, bound to the enzyme. The proposal will also (2) quantitatively study the inhibition of erythrocyte G6PD kinetics by ATP and 2,3 diphosphoglycerate, two proposed physiologically important inhibitors of G6PD. The third (3) specific aim will measure the dimertetramer association constant using large zone (plateau) HPLC experiments on a size exclusion column.