Kinetic analysis of the amino terminal end of active site loop of lactate deyhdrogenase from plasmodium vivax

Amaç: Bu çalışmada aktive bölge amino terminal ucundaki amino asitlerin fonksiyonel öneminin anlaşılması için önceden Toxoplasma gondii I, II, Eimeria acervulina ve Eimeria tenella LDH enzimlerine benzetilmiş olan Plasmodium vivax LDH mutant enzimlerinin kinetik analizleri gerçekleştirilmiştir. Yöntemler: PZR aracılığı ile mutant genler çoğaltılarak enzimlerin C terminalinde 6xHistag ifade edilmesi sağlanmıştır. Mutant LDH enzimleri E. coli hücrelerinde rekombinant olarak üretilmiş, Ni-NTA agaroz kolonları kullanılarak saflaştırılmış ve mutant enzimlerin kinetik özellikleri belirlenmiştir. Bulgular: Mutant enzimlerin Km pürivat değerlerinde önemli bir artışın gözlemlenmiş olması bu bölgede yapılan değişikliklerin enzimin substratına olan ilgisini belirgin bir şekilde düşürmesine neden olduğunu göstermiştir. Fakat kcat pürivat değeri bütün proteinlerde hemen hemen aynı kalmıştır. Sonuç: Söz konusu bölgenin hassasiyeti, bu bölgenin ilaç tasarım çalışmaları için gerek Plasmodium gerek diğer Apicomplexan’lar için önemini vurgulamaktadır.

Plasmodium vivax laktat dehidrogenaz enziminin (PvLDH) aktif bölge amino terminal ucunun kinetik analizi

Objective: In this study, kinetic analysis was performed to understand the functional importance of the amino terminal of the active site of previously mutated Plasmodium vivax Lactate Dehydrogenase enzyme by mimicking Toxoplasma gondii I, II, Eimeria acervulina and Eimeria tenella LDH's. Material and Methods: Mutant LDH genes were amplified by PCR and 6xHistag was added to the C-terminal of the enzymes. Then LDH enzymes are overproduced as recombinant in E. coli cells, purified by Ni-NTA agarose matrix and kinetic properties were analysed. Results: Observing increase of Km values of mutant enzymes showed that mutations in this place caused decreasing affinity of enzyme for its substrate. However kcat values were about the same throughout all mutant proteins. Conclusion: Sensitivity of the studied region emphasizes the significance of this site for drug design studies for both Plasmodium and some other Apicomplexans.

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1. Knell AJ. Malaria, Oxford; Oxford University Press: 1991.
2. Cox-Singh J, Davis TM, Lee KS, Shamsul SS, Matusop A, Ratnam S, et al. Plasmodium knowlesi Malaria in humans is widely distributed and potentially life threatening. Clin Infect Dis 2008;46:165-71. [CrossRef]
3. World Health Organization (WHO). The global burden of disease: 2004 update. Switzerland: WHO Press; 2008.
4. Patel AP, Staines HM, Krishna S. New antimalarial targets: The example of glucose transport. Travel Med Infect Dis 2008;6:58- 66. [CrossRef]
5. Baird JK. Chloroquine resistance in Plasmodium vivax. Antimicrob Agents Chemother 2004;48:4075-83. [CrossRef]
6. Sherman IW. Biochmesitry of Plasmodium (malarial parasites). Microbiol Rev 1979;43:453-95.
7. Basco LK, Marquet F, Makler MM, Le Bras J. Plasmodium falciparum and Plasmodium vivax: Lactate dehydrogenase activity and its application for in vitro drug susceptibility assay. Exp Parasitol 1995;80:260-71. [CrossRef]
8. Royer RE, Deck LM, Campos NM, Hunsaker LA, Vander Jagt DL. Biologically active derivatives of gossypol: synthesis and antimalarial activities of peri-acylated gossylic nitriles. J Med Chem 1986;29:1799-801. [CrossRef]
9. Vivas L, Easton A, Kendrick H, Cameron A, Lavandera JL, Barros D, et al. Plasmodium falciparum: stage specific effects of a selective inhibitor of lactate dehydrogenase. Exp Parasitol 2005;111:105-14. [CrossRef]
10. Dunn CR, Banfield MJ, Barker JJ, Higham CW, Moreton KM, Turgut- Balik D, et al. The structure of lactate dehydrogenase from Plasmodium falciparum reveals a new target for anti-malarial design. Nat Struct Bio 1996;11:912-5. [CrossRef]
11. Gomez MS, Piper RC, Hunsaker LA, Royer RE, Deck LM, Makler MT, et al. Substrate and cofactor specificity and selective inhibition of lactate dehydrogenase from the Malarial parasite P. falciparum. Mol Biochem Parasitol 1997;90:235-46. [CrossRef]
12. Turgut-Balık D, Holbrook JJ. Determination of the DNA and amino acid sequences of the lactate dehydrogenasegene from Plasmodium falciparum, strains K1 and PF FCBR: A route to the design of new antimalarials. Turk j Biol 2001;25:241-50.
13. Turgut-Balik D, Shoemark DK, Sessions RB, Moreton KM, Holbrook JJ. Mutagenic exploration of the active site of lactate dehydrogenase from Plasmodium falciparum. Biotech Lett 2001;23:923-7. [CrossRef]
14. Turgut-Balik D, Shoemark DK, Moreton KM, Sessions RB, Holbrook JJ. Over-production of lactate dehydrogenase from Plasmodium falciparum opens route to new antimalarials. Biotech Lett 2001;23:917-21. [CrossRef]
15. Turgut-Balık D, Akbulut E, Shoemark DK, Celik V, Moreton KM, Sessions RB, et al. Cloning, sequence and expression of the lactate dehydrogenase gene from human malaria parasite Plasmodium vivax. Biotech Lett 2004;26;1051-5. [CrossRef]
16. Sambrook J, Russell DW. Molecular cloning a laboratory manual I-II-III. New York: CSHL Pres; 2001.
17. Atmıs B. Plasmodium vivax’ ın LDH enziminin aktif bölge halkasının amino terminal ucunun Analizi. Master of Science Thesis; Department of Biology; University of Firat; 2007.
18. Turgut-Balık D, Çelik V, Moreton KM, Brady RL. Overcoming cloning problems by staining agarose gels with crystal violet ınstead of ethidium bromide in lactate dehydrogenase gene from Plasmadium vivax and Plasmodium falciparum. Acta Biol Hung 2005;56:389-97. [CrossRef]
19. Shoemark DK. The kinetic characterization of the lactate dehydrogenase enzyme from Plasmodium falciparum. UK: Doctor of philosophy thesis; Department of biochemistry; University of Bristol; 2000.
20. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;5259:680-5. [CrossRef]
21. Leatherbarrow RJ. (Grafit, Version 3.0.): Staines. UK: Erithracus Software Ltd; 1992.
22. Berwal R, Gopalan N, Chandel K, Prasad GB, Prakash S. Plasmodium falciparum: enhanced soluble expression, purification and biochemical characterization of lactate dehydrogenase. Exp Parasitol 2008;120:135-41. [CrossRef]
23. Chaikuad A, Fairweather V, Conners R, Josep-Horne T, Turgut- Balik D, Brady RL. Structure of Lactate Dehydrogenase from Plasmodium vivax: Complexes with NADH and APADH. Biochemistry 2005;44:16221-8. [CrossRef]
24. Mutlu O. Plasmodium vivax yabanıl tip laktat dehidrogenaz enziminin saflaştırılmasının optimizasyonu ve yabanıl tip ile mutant enzimlerinin kinetik analizlerinin yapılması. Master of Science Thesis; Department of Bioengineering; Yildiz Technical University; 2009.