RESUMO
We report an assay for the determination of the activity of 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase, the enzyme which catalyzes the fourth reaction step of the 2-C-methyl-D-erythritol 4-phosphate pathway for the synthesis of isoprenoids, which is based on the spectrophotometrical determination of adenosine 5'-diphosphate using pyruvate kinase and L-lactate dehydrogenase as auxiliary enzymes. This method can be adapted to microtiter plates, can be automated, and because of its simplicity and speed can be useful for the functional characterization of the enzyme and for the screening of inhibitors with potential antibiotic or antimalarial action.
Assuntos
Proteínas de Escherichia coli/análise , Escherichia coli/enzimologia , Fosfotransferases (Aceptor do Grupo Álcool)/análise , Espectrofotometria/métodos , Difosfato de Adenosina/análise , Automação , Cromatografia Líquida de Alta Pressão , Inibidores Enzimáticos/isolamento & purificação , Cinética , L-Lactato Desidrogenase/metabolismo , Microquímica , NAD/metabolismo , Fosfoenolpiruvato/metabolismo , Piruvato Quinase/metabolismoRESUMO
A new method for the determination of the activity of 4-diphosphocytidyl-2-C-methyl-D-erythritol 4-phosphate synthase, the enzyme catalyzing the third reaction of the 2-C-methyl-D-erythritol 4-phosphate pathway for biosynthesis of isoprenoids, is described. This is an end-point assay based on the transformation of inorganic pyrophosphate, one of the products of the reaction, to phosphate by using inorganic pyrophosphatase as auxiliary enzyme. The phosphate formed is reacted then with the dye malachite green to yield a colored product which can be determined spectrophotometrically. The method is easy to perform, sensitive, and robust and can be used in automated high-throughput screening analyses for the search of inhibitors of the enzyme.
