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Journal of Biochemistry Advance Access published online on May 13, 2008
Journal of Biochemistry, doi:10.1093/jb/mvn062
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© 2008 The Japanese Biochemical Society

Molecular characterization, heterologous expression and kinetic analysis of recombinant Plasmodium falciparum thymidylate kinase

Mahmoud Kandeel1 and Yukio Kitade1,2,3,4,*

1Department of Biomolecular Science, Faculty of Engineering, 2Center for Advanced Drug Research, 3Center for Emerging Infectious Diseases,4United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University, Yanagido 1-1, Gifu 501-1193, Japan

*To whom correspondence should be addressed. Prof. Yukio Kitade. Tel./fax: +81 58 293 2640, E-mail: ykkitade{at}gifu-u.ac.jp

Received April 24, 2008; Accepted May 1, 2008


  Abstract

The gene encoding for thymidylate kinase from Plasmodium falciparum was obtained by PCR and expressed in Escherichia coli and the enzyme was investigated as a possible new drug target. The enzyme is a homodimer exhibiting maximal kinase activity over a wide pH range of 7-9 and is characterized by marked stability. Compared with the human enzyme, the recombinant Plasmodium falciparum TMP kinase showed a broader spectrum of substrate specificity. The enzyme not only phosphorylates dTMP and dUMP but can also tolerate the bulkier purines dGMP, GMP and dIMP. Initial velocity studies showed that the Km values for TMP and dGMP are 22 and 30 µM, respectively. The turnover number kcat(TMP) was found to be 3.4 s-1, a value indicating the higher catalytic efficiency of the plasmodium enzyme. From the present study we suggest that the design of appropriate inhibitors especially purine based compounds could have a selective inhibitory effect on the parasite enzyme.

Key Words: thymidylate kinase, Plasmodium falciparum, malaria, enzyme kinetics


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