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Phosphoglucose isomerase (EC5.3.1.9) catalyzes the inter-conversion between D-glucopyranose 6-phosphate and D-fructofuranose 6-phosphate by promoting an intra-hydrogen transfer between C1 and C2 A base has been proposed to initate the transfer of hydrogen (Dyson & Noltmann, 1968; Rose & O'' connell, 1961; Schray et al, 1973; Malaisse et al 1990). A conserved histidine has been found throughout all phosphoglucose isomerases, and proposed to be the base catalyzing the isomerization reaction. In this study the conserved histidine, His311, of the enzyme from Bacillus stearothermophilus was studied by sitedirected mutagenesis to evaluate its possibility of being a base. His31, His32, and His311 were substituted with alanine individually. The kinetic analysis showed that the activity of H311A decreased to 0.04% of that of wild-type enzyme, while H31A and H32A remained unchanged in their activites His311 was further changed to asparagine and glutamine. The catalytic efficiency, Kcat/Km2 of these two mutant enzymes were similar to that of H311A, suggesting that His311 functions as a base instead of a hydrogen-bond donor or acceptor. N-bromoacetylethanolamine phosphate, an active-site directed inhibitor, inhibits irreversibly the activity of wild-type phosphoglucose isomerase. However, H311A became resistant to the inhibiton of N-bromoacetylethanolamine phosphate indicationg that His311 is really located in the active site of the enzyme. The result of the pH dependence of Kcat for the enzyme also suggests that His311 functioned as a general base in the catalysis.
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