A spectrophotometric assay for the determination of 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase activity.
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.
Bernal
Cristobal
C
Departament de Bioquimica i Biologia Molecular, Universitat de Barcelona, Avda Diagonal 645, 08028 Barcelona, Spain.
Mendez
Eva
E
Terencio
José
J
Boronat
Albert
A
Imperial
Santiago
S
D000244:Adenosine Diphosphate / Q000032:analysis; D001331:Automation; D002851:Chromatography, High Pressure Liquid; D004791:Enzyme Inhibitors / Q000302:isolation & purification; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000032:analysis*; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008832:Microchemistry; D009243:NAD / Q000378:metabolism; D010728:Phosphoenolpyruvate / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000032:analysis*; D011770:Pyruvate Kinase / Q000378:metabolism; D013053:Spectrophotometry / Q000379:methods*
D004791:Enzyme Inhibitors; D029968:Escherichia coli Proteins; D009243:NAD; D000244:Adenosine Diphosphate; D010728:Phosphoenolpyruvate; D007770:L-Lactate Dehydrogenase; D017853:Phosphotransferases (Alcohol Group Acceptor); C404502:IspE protein, E coli; D011770:Pyruvate Kinase