| Summary: | 碩士 === 國立中興大學 === 農業生物科技學研究所 === 90 === . Phosphoglucose isomerase （E C 5.3.1.9;PGI） is a dimeric enzyme that catalyzes the reversible inter-conversion of glucose-6-phosphate（G-6-P）and fructose-6-phosphate（F-6-P） in glycolysis and gluconeogenesis pathways. Phosphoglucose isomerase deficiency is the third most common enzyme deficiency known to cause hereditary nonspherocytic hemolytic anemia. Our aim is to analyze the catalytic efficiency and structural stability of the inherited mutant phosphoglucose isomerase. The mutated proteins were generated by site-directed mutagenesis and expressed in E. coli. The activity assays show that mutant proteins R83W、V101M、R347C and R472H decrease approximately 2 folds, R347H decreases approximately 3 folds, and I524T decreases approximately 20-fold compare with the kcat/KM of wide type enzyme. R273H has no activity even a large amount of the enzyme was used for activity assay. S278L and E495K could not be obtained under the same purification conditions suggesting that they have problem in protein folding. Regarding thermostability, the half-lives (t1/2) of mutated proteins were much less stable than WT event at saturated concentration of substrate. I524T is slightly unstable compared with wild-type enzyme at 55 ℃ with 1 mM F-6-P. Combined with the 3-D structure of human phosphoglucose isomerase, I also attempt to address the reason causing the decrease of thermostability of each mutant PGI protein. More hereditary mutated PGI proteins will be characterized in the future to address the structure-function relationship of the hereditary mutations of phosphoglucose isomerase.
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