探討人類葡萄糖-六-磷酸去氫脢上的八個半胱胺基酸在蛋白結構與功能上的角色

葡萄糖-六-磷酸去氫酵素 (Glucose-6-phoshate dehydrogenase; G6PD) 是葡萄糖經由六碳糖磷酸分路 (Hexose Monophospate Shunt) 代謝的第一個主要酵素。在這一個途徑之中，其主要的生化產物為NADPH和Ribose-5-phosphate，而其中NADPH是保護細胞不受氧化物質損傷 (oxidative damage) 的重要還原物質。 而葡萄糖-六-磷酸去氫酵素缺乏症(Glucose-6-phoshate dehydrogenase deficiency) 是世界上最常見的酵素缺乏疾病。跟據統計，全世界目前大約有兩億多人因為此酵素缺乏而受到影響。由於近年來分子生物學的技術發達，我們發現人類的G6PD缺乏症大都是由於其基因中核酸的點突變所造成的。而G6PD基因也已經被證實是位於人類的X染色體長臂上，且整個基因序列也被定序出。 跟據定序出的核酸序列發現，整個G6PD基因是由515個胺基酸所構成，可是至今整個G6PD蛋白上的個別胺基酸對於此蛋白的功能和結構上所產生的影響尚未知。在本論文的研究中，我們對整個G6PD基因上的八個半胱胺酸 (Cysteine) 以體外定點突變 (in vitro site-directed mutagenesis) 的方式和其他它相關分子生物學技術，將這八個半胱胺酸分別置換成甘胺酸 (Glycine) 和絲胺酸 (Serine) 。這些突變後的基因，利用核酸定序的方法加以證實，並使用基因重組的方法，再將個別不同突變的G6PD基因植入載體，並送入G6PD缺陷型的大腸桿菌HB351(DE3)中成為一完整的G6PD蛋白表達系統，以IPTG誘導產生G6PD蛋白，即可用來進行G6PD酵素活性的測試。 實驗結果顯示，這八個半胱胺酸可以分成五個類型: 第一是Cys358，此種突變對G6PD的活性沒有太大的影響。第二類型為Cys13、Cys158、Cys385，此位置的cysteine的供能可能為提供蛋白結構上電親合力。第三類是Cys269，當cysteine被取代為serine時對活性有明顯的影響但取代成glycine則否，可能與NADP的結合有關。第四類型為Cys232、Cys446，不管是取代為serine或glycine，其活性變得非常低或沒有活性，可能和G6PD蛋白雙合體(Dimer)間或是單體間的雙硫鍵有關。第五類型是Cys294，當胺基酸被取帶為glycine的活性比取代為serine來的好，但此結果還要更進一步的實驗來證明才可得知。

Glucose-6-phosphate dehydrogenase (G6PD) is a key enzyme in the hexose monophosphate shunt and its major biochemical function is to generate NADPH, an important reducing element to prevent cell from oxidative damages. Molecular biological studies of the G6PD deficiency have revealed that the location of the gene in the long arm region of X chromosome and the cDNA has been cloned and sequenced. The deduced amino acid sequences from its DNA sequences have revealed that G6PD contains 515 amino acid. However, little is known about the functional roles of those amino acid residues of G6PD protein. Especially those of the unique amino acids, such as cysteine, have never been fully studied. In this study, using site-directed mutagenesis techniques, each of the eight different cysteines in G6PD protein has been successfully mutated to either serine or glycine. Mutants were confirmed by DNA sequencing and mutant proteins were expressed in the G6PD deficient E. coli strain HB351(DE3). The cell lysates were assayed for enzyme activity after a proper induction. Based on the results from this study, we found that the functions of these cysteine residues in human G6PD can be categorized into 5 different groups. The first group includes the cysteine residue at the amino acid position 358, in which cysteine residue might not be important for the protein function. The second group includes the cysteine residues at positions 13, 158, and 385 of G6PD protein. The serine substitutions of these cysteines possibly provide a slight better electrostatics environment for activity than those of glycine substitutions. The third group includes the cysteine 269 in G6PD. The electrophilic feature of the G6PD protein at this position might be important for binding to its cofactor, namely the NADP+. The fourth group includes the cysteines 232 and 446 in G6PD. These cysteine residues might form the inter/intra- molecular disulfide bonds and, therefore, both are important for their structural roles in the G6PD protein. The fifth group includes the cysteines 294 in G6PD, however, its role in G6PD activity still needs to be clarified.