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-磷酸脱氢酶（G6PD）的动力学和热力学研究，该研究涉及控制酶的结合状态。G6PD是六糖膦途径的关键酶，目前已鉴定出370多种人类红细胞酶的遗传变异。G6PD缺乏症患者在接触多种药物或食物时会出现溶血危机。大量的遗传变异，许多活性降低，使G6PD成为克隆和测序的一个有吸引力的目标。氨基酸序列变化的解释依赖于G6PD生化特性的测定。不幸的是，对红细胞G6PD的许多基本酶特性，即底物的结合和细胞间分子对酶动力学的影响，缺乏一致意见。与之前对酵母G6PD的研究类似，本研究的假设是过去对人红细胞G6PD的动力学和热力学研究，测量了G6PD二聚体和四聚体混合物的性质，每种G6PD可能具有不同的生化性质。由于G6PD二聚体和四聚体的相对数量在动力学或热力学测量过程中发生了变化，因此在人红细胞G6PD的生化特性方面缺乏一致性。因此，建议将重点放在测量G6PD二聚体的生化特性上，这种形式被认为存在于红细胞中。RUI提案的三个具体目的是：(1)测量与G6PD结合的烟酰胺腺嘌呤二核苷酸磷酸（NADP）和葡萄糖-6-磷酸（G6P）分子的数量以及这两个分子的结合常数的大小。该项目将直接测量与酶结合的放射性标记G6P或NADP的平衡数量。该提案还将(2)定量研究ATP和2,3二磷酸甘油酸对红细胞G6PD动力学的抑制作用，这两种被提出的G6PD生理上重要的抑制剂。第三(3)具体目标将测量二聚体结合常数使用大区域（平台）高效液相色谱实验的尺寸排除柱。

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.