| Summary: | 碩士 === 國立清華大學 === 生物科技研究所 === 94 === Bacterial UMP kinases are important and essential enzymes which are involved in the multistep synthesis of nucleoside triphosphates. The known homohexamer from bacteria with no similar with eukaryotic organisms, might be a target for designing new antibacterial drugs. In this study, the HP0777 gene encoding UMP kinase in Helicobacter pylori strain 26695 was overexpressed. This protein has a molecular mass of 27.56 kD, and the Isoelectric point (pI) determination of 8.67.
The recombinant HP0777 has low solubility at pH = 8 (≦ 0.5ug of protein/ul), but its solubility can be increased at an alkaline pH of 9. Rec-HP0777 showed UMP kinase activity in the presence of 2 mM MgCl2. The maximum UMP kinase activity of this protein appeared in reaction at 30 oC. About 10% enzyme activity remained after recombinant protein kept at 65 oC for 15 min and assayed at 30 oC.
UMP kinase activity of rec-HP0777 protein was regulated by the activator GTP and inhibited by UTP. GTP at 0.15 mM could increase more than fifteen folds enzyme activity of rec-HP0777 protein, in contrast, 1 mM GTP increased 3-4 folds UMP kinase activity in E. coli from known published data. UTP at 1 mM decreased 70% of the UMP kinase activity for rec-HP0777 protein.
UMP kinase activity of rec-HP0777 protein increased twice when magnesium was replaced with manganese at 2 mM. The activity reduced to 50%, 7% while cobalt or nickel ions were applied into reaction. When calcium or copper ions were substituted for manganese ions in the reaction mixture, the enzyme activity was not detectable. Determination of the enzyme activity at different pH value (6.5-9.5) indicated that the maximum UMP kinase activity of rec-HP0777 protein appeared in reaction buffer at pH 9.0 in present of magnesium and at pH 7.4 in present of manganese.
Although the amino acid sequence of HP0777 shares almost 50% identity and 70% similarity to several known UMP kinase protein sequence from bacteria, the low concentration of GTP activator and manganese preference instead of magnesium for UTP kinase activity are novel discovery for HP0777 protein.
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