Escherichia coli isocitrate dehydrogenase kinase/phosphatase. Overproduction and kinetics of interaction with its substrates by using intrinsic fluorescence and fluorescent nucleotide analogues.

摘要: The aceK gene of Escherichia coli, which encodes the isocitrate dehydrogenase kinase/phosphatase (IDH K/P), was cloned in pQE30 expression vector to overproduce a protein tagged with six histidine residues at its N-terminus. By using one-step chromatographic procedure, IDH K/P purified near homogeneity. K/P, contains nine Trp residues, exhibited characteristic intrinsic tryptophan fluorescence low maximal emission 326 nm. value Stern-Volmer quenching constant presence acrylamide (Ksv = 2.1 M-1) indicated that were deeply buried protein. Furthermore, very sensitive binding nucleotide. induced by nucleotide together an increased fluorescent analogues, methylanthraniloyl-derivatives ADP, ATP, GDP and GTP adenosine-5'-triphosphoro-1-(5-sulfonic-acid) naphthylamidate, used investigate interaction K/P. dimer shown contain two identical sites, one on each subunit, Kd range 1.7-2.5 microM for unmodified ADP or ATP 2.5-3.7 fluorescently labelled nucleotides. In contrast, affinity 10-fold lower than adenine site located within 315-340 limited proteolysis endoproteinase Lys-C. Only main cleavage obtained: peptide bond K346-E347 strongly protected ATP.