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Title: Mutational analysis of Plasmodium falciparum dihydrofolate reductase: The role of aspartate 54 and phenylalanine 223 on catalytic activity and antifolate binding
Authors: Worachart Sirawaraporn
Rachada Sirawaraporn
Suganya Yongkiettrakul
Amornpol Anuwatwora
Guilio Rastelli
Sumalee Kamchonwongpaisan
Yongyuth Yuthavong
Mahidol University
Thailand National Center for Genetic Engineering and Biotechnology
Universita degli Studi di Modena e Reggio Emilia
Keywords: Biochemistry, Genetics and Molecular Biology;Immunology and Microbiology
Issue Date: 18-Jun-2002
Citation: Molecular and Biochemical Parasitology. Vol.121, No.2 (2002), 185-193
Abstract: The catalytic activity and ability to confer resistance to antifolates of Plasmodium falciparum dihydrofolate reductase (pfDHFR) through single and double mutations at Asp-54 and Phe-223 were investigated. A single Asp54Glu (D54E) mutation in the pfDHFR domain greatly decreased the catalytic activity of the enzyme and affected both the Kmvalues for the substrate dihydrofolate and the Kivalues for pyrimethamine, cycloguanil and WR99210. The Phe223Ser (F223S) single mutant had unperturbed kinetics but had very poor affinity with the first two antifolates. The ability to confer high resistance to the antifolates of F223S enzyme was, however, abolished in the D54E+F223S double mutant enzyme. When D54E mutation was present together with the A16V+S108T double mutation, the effects on the Kmvalues for the substrate dihydrofolate and the binding affinity of antifolates were much more pronounced. The severely impaired kinetics and poor activity observed in A16V+S108T+D54E enzyme could, however, be restored when F223S was introduced, while the binding affinities to the antifolates remained poor. The experimental findings can be explained with a model for substrate and inhibitor binding. Our data not only indicate the importance of Asp-54 of pfDHFR in catalysis and inhibitor binding, but also provide evidence that infer the potentially crucial function of the C-terminal portion of pfDHFR domain. © 2002 Elsevier Science B.V. All rights reserved.
ISSN: 01666851
Appears in Collections:Scopus 2001-2005

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