Molecular characterization and mutational analysis of recombinant diadenosine 5',5″-P¹,P⁴-tetraphosphate hydrolase from Plasmodium falciparum.
Asymmetrical diadenosine 5',5"-P¹,P⁴-tetraphosphate hydrolase (EC 3.6.1.17) from human malaria parasite Plasmodium falciparum was expressed in Escherichia coli, purified to homogeneity, and characterized for the first time as a biological target for chemotherapeutic agents against malaria. Plasmodium falciparum Ap₄A (PfAp₄A) hydrolase not only catalyzes diadenosine 5',5″-P¹,P⁴-tetraphosphate (Ap₄A) to ATP and AMP, but also diadenosine 5″-P¹,P⁵-pentaphosphate (Ap₅A) to ATP and ADP. Marked enzyme heat stability corresponding to the highest level of activity was observed at 60°C. The recombinant enzyme showed maximal activity in the presence of 5 mM Mg²⁺ ions. Kinetic analysis revealed the values of K(m) and K(cat) as 0.6 μM and 2.5 min⁻¹, respectively. Comparative protein modeling indicated an additional space in the substrate binding site of the parasitic enzyme compared with that of humans. Mutagenic analysis of the amino acid residue (Pro133) forming the additional space revealed a 5-fold increase in the wild-type Km value when replaced by a smaller (Ala) residue. Furthermore, catalytic activity was markedly affected by introducing a larger residue (Phe), thus creating the potential to develop a specific inhibitor of PfAp₄A hydrolase.
United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University, Japan.
Waleed
W
Osman
Satoshi
S
Endo
Kentaro
K
Oh-Hashi
Yoshiaki
Y
Kitamura
Yukio
Y
Kitade
D017766:Acid Anhydride Hydrolases / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D004252:DNA Mutational Analysis; D015226:Dinucleoside Phosphates / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010963:Plasmodium falciparum / Q000201:enzymology*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment
D015226:Dinucleoside Phosphates; D011994:Recombinant Proteins; C020733:diadenosine tetraphosphate; D017766:Acid Anhydride Hydrolases; C020023:bis(5'-nucleosyl)tetraphosphatase (asymmetrical)