| Summary: | 碩士 === 中山醫學院 === 醫學研究所 === 88 === 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.
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