Investigating the binding site of Human Phosphoglucose Isomerase for GTP

• Main Authors: Ka-Lik Cheng, 鄭加力
• Other Authors: Menghsiao Meng
• Format: Others
• Language: zh-TW
• Published: 2015
• Online Access: http://ndltd.ncl.edu.tw/handle/16560477132078822774

碩士 === 國立中興大學 === 生物科技學研究所 === 103 === Phosphoglucose isomerase (PGI) is a protein with multiple functions. It catalyzes the interconversion between glucose 6-phosphate (G6P) and fructose 6-phosphate (F6P). Widely existing in diverse species, PGI acts as an isomerization enzyme in glycolysis and gluconeogenesis pathways. Guanosine triphosphate (GTP) is used as a source of energy for protein synthesis and gluconeogenesis. GTP also played a role as an activator of substrates in metabolic reactions. In a precious study, human PGI was found to interact with GTP, but not with Escherichia coli (E. coil) PGI. The purpose of this study is to investigate which amino acids on the human PGI will interact with GTP and to find out the consequence of the interaction. First of all, hPGI could hydrolyzed GTP when the MgCl2 include in the buffer. Several phosphosugar were used to interfere with the UV-induced [α-32P]-GTP crosslink to human PGI. The results showed that GTP, F6P and G6P could affect the crosslink between [α-32P]-GTP and the protein. Enzyme kinetic studies indicated that both of GTP and 6-phosphate gluconate (6P-GA) are competitive inhibitors with the values of Ki 63 μM and 12 μM, respectively. Yonetani-Theorell double-inhibition experiment found that both GTP and 6P-GA have values of α approximately 1.7 ；suggesting that GTP and 6P-GA could interfere with each other for the binding to human PGI. The previous study also suspected that the 92 tyrosine (Tyrosine, Y) and 95 glycine (Glycine, G) residues may involve the GTP binding. Therefore, mutations of Y92R, G95N and Y92R/G95N were created; on the other hands, mutations of R90Y, N93G and R90Y/N93G in E. coli PGI were created. GTP-binding assay indicating that Y92R/G95N weakens the binding of GTP to human PGI. The GTP binding specified area of hPGI investigating by molecular modelling prediction estimated the minimum Gibbs free energy about -10.7 kcal/mol. R96 and Q512 are simultaneously in the three predicted structure. In the protein crystallization experiments with three crystallization conditions, hPGI protein crystals were collected, but the GTP combination with human PGI was not found. X-ray results showed a similar phosphate structures embedded in the S159, S209, K210, T214 and H288.