Kinetic Analysis of the Amino Terminal End of Active Site Loop of Lactate Deyhdrogenase from Plasmodium Vivax

Abstract

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. Turkish Başlık: Plasmodium vivax Laktat Dehidrogenaz Enziminin (PvLDH) Aktif Bölge Amino Terminal Ucunun Kinetik Analizi Anahtar Kelimeler: Laktat dehidrogenaz, Plasmodium vivax, Antimalarial ilaç, Yönlenlendirilmiş mutagenez, Kinetik analiz 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.

Keywords

• Lactate dehydrogenase, Plasmodium vivax, antimalarial drug, site-directed mutagenesis, kinetic analysis

Kinetic Analysis of the Amino Terminal End of Active Site Loop of Lactate Deyhdrogenase from Plasmodium Vivax

Öz

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