Bioinformatics, enzymologic properties, and comprehensive tracking of Plasmodium falciparum nucleoside diphosphate kinase.
The gene encoding for nucleoside diphosphate kinase from Plasmodium falciparum was obtained by polymerase chain reaction (PCR) and expressed in Escherichia coli. Tracking kinases is strenuous work due to many functional and technical deficits. Tracking of Plasmodium falciparum nucleoside diphosphate kinase (PfNDK) was carried out by conventional enzyme assays combined by isothermal titration calorimetry (ITC). ITC proved an efficient tracking method with rapid, accurate, and confident target confirmation. In addition, it provides substrate affinity and full thermodynamic profile in one experiment. Magnesium ions were found to be essential for nucleoside diphosphate (NDP) kinase activity; however, the absence of Mg(2+) did not completely interfere with the binding of nucleotides. The substrate recognition was found to depend on enthalpic forces with little entropic contributions. However, in the absence of magnesium ions the nucleotides actively bind to the enzyme driven by hydrophobic forces. The enzyme showed specific activity that was within the average of known enzymes; however, it was at least two-fold higher than that of the human enzyme.
Kandeel
Mahmoud
M
Department of Biomolecular Science, Faculty of Engineering, Gifu University, Gifu, Japan.
Miyamoto
Tatsuya
T
Kitade
Yukio
Y
D001483:Base Sequence; D002151:Calorimetry; D019295:Computational Biology; D017124:Conserved Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009701:Nucleoside-Diphosphate Kinase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D009705:Nucleosides / Q000737:chemistry / Q000494:pharmacology; D010957:Plasmids; D010963:Plasmodium falciparum / Q000201:enzymology* / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013379:Substrate Specificity; D013816:Thermodynamics; D017186:Titrimetry
D009705:Nucleosides; D009701:Nucleoside-Diphosphate Kinase