Mutational, inhibitory and microcalorimetric analyses of Plasmodium falciparum TMP kinase. Implications for drug discovery.
Plasmodium falciparum thymidylate kinase (PfTMK) can tolerate a range of substrates, which distinguishes it from other thymidylate kinases. The enzyme not only phosphorylates TMP and dUMP but can also tolerate bulkier purines, namely, dGMP, GMP, and dIMP. In order to probe the flexibility of PfTMK in accommodating ligands of various sizes, we developed 6 mutant enzymes and subjected these to thermodynamic, inhibitory and catalytic evaluation. Kinase activity was markedly affected by introducing a larger lysine residue instead of A111. The lack of the hydroxyl group after inducing mutation of Y107F affected enzyme activity, and had a more severe impact on dGMP kinase activity. PfTMK can be inhibited by both purine and pyrimidine nucleosides, raising the possibility of developing highly selective drugs. Thermodynamic analysis revealed that enthalpic forces govern both purine and pyrimidine nucleoside monophosphate binding, and the binding affinity of both substrates was highly comparable. The heat produced due to dGMP binding is lower than that attributable to TMP. This indicates that additional interactions occur with TMP, which may be lost with larger dGMP. Targeting PfTMK not only affects thymidine nucleotide synthesis but may also affect purine nucleotides, and thus the enzyme represents an attractive antimicrobial target.
Kandeel
M
M
Department of Biomolecular Science, Faculty of Engineering, Gifu University, Yanagido 1-1, Gifu 501-1193, Japan.
Ando
T
T
Kitamura
Y
Y
Abdel-Aziz
M
M
Kitade
Y
Y
D000818:Animals; D002151:Calorimetry; D055808:Drug Discovery; D004789:Enzyme Activation / Q000187:drug effects; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009703:Nucleoside-Phosphate Kinase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009705:Nucleosides / Q000737:chemistry / Q000494:pharmacology; D010963:Plasmodium falciparum / Q000201:enzymology*; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary; D013816:Thermodynamics
D009705:Nucleosides; D009703:Nucleoside-Phosphate Kinase; C020093:dTMP kinase