Molecular cloning, expression, characterization and mutation of Plasmodium falciparum guanylate kinase

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• 作者：Mahmoud K ; eel ; Masayuki Nakanishi ; Takayuki Ando ; Kamal El-Shazly ; Tarek Yosef ; Yoshihito Ueno ; Yukio Kitade
• 关键词：Plasmodium falciparum ; Guanylate kinase ; Site-directed mutagenesis ; Drug targets
• 刊名：Molecular and Biochemical Parasitology
• 出版年：2008
• 出版时间：June 2008
• 年：2008
• 卷：159
• 期：2
• 页码：130-133
• 全文大小：481 K

文摘

The present work describes cloning, expression, purification, characterization, and mutation of Plasmodium falciparum guanylate kinase (PlasmoDB ID PFI1420w). Amino-acid sequence alignment revealed important differences especially in K42-V51, Y73-A77, and F100-L110, which include residues important for kinase activity, and at helix 3, which is important for domain movements. The catalytic efficiency for dGMP was 22-fold lower than that for GMP, whose value is the lowest among known guanylate kinases. dGMP was found to a competitive inhibitor for GMP with K_i = 0.148 mM and a mixed-type inhibitor with regard to ATP with measured K_i = 0.4 mM. The specificity constant (K_cat/K_m) of the four examined mutants varied for natural substrate GMP/dGMP, indicating the involvement of different mechanisms in substrate recognition and subsequent loop-domain movement. These results show that P. falciparum guanylate kinase is structurally and biochemically distinct from other guanylate kinases and could be a possible target in drug development.