Escherichia coli isocitrate dehydrogenase kinase/phosphatase. Overproduction and kinetics of interaction with its substrates by using intrinsic fluorescence and fluorescent nucleotide analogues.
The aceK gene of Escherichia coli, which encodes the isocitrate dehydrogenase kinase/phosphatase (IDH K/P), was cloned in the pQE30 expression vector to overproduce a protein tagged with six histidine residues at its N-terminus. By using a one-step chromatographic procedure, the IDH K/P was purified to near homogeneity. The IDH K/P, which contains nine Trp residues, exhibited a characteristic intrinsic tryptophan fluorescence with a low maximal emission at 326 nm. The low value of the Stern-Volmer quenching constant in the presence of acrylamide (Ksv = 2.1 M-1) indicated that the tryptophan residues were deeply buried in the protein. Furthermore, the intrinsic tryptophan fluorescence was very sensitive to the binding of nucleotide. The quenching of protein fluorescence induced by the binding of nucleotide together with an increased intrinsic fluorescence of fluorescent nucleotide analogues, methylanthraniloyl-derivatives ADP, ATP, GDP and GTP and adenosine-5'-triphosphoro-1-(5-sulfonic-acid) naphthylamidate, were used to investigate the interaction with IDH K/P. The IDH K/P dimer was shown to contain two identical nucleotide binding sites, one on each subunit, with a Kd in the range of 1.7-2.5 microM for unmodified ADP or ATP and of 2.5-3.7 microM for fluorescently labelled nucleotides. In contrast, the affinity for GDP or GTP was 10-fold lower than for adenine nucleotides. The nucleotide binding site was located within residues 315-340 by using limited proteolysis of IDH K/P by endoproteinase Lys-C. Only one main site of cleavage was obtained: the peptide bond K346-E347 which was strongly protected in the presence of ATP.
Rittinger
K
K
Max-Planck-Institut für Medizinische Forschung, Abteilung Biophysik, Heidelberg, Germany.
Negre
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D
Divita
G
G
Scarabel
M
M
Bonod-Bidaud
C
C
Goody
R S
RS
Cozzone
A J
AJ
Cortay
J C
JC
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology*; D005453:Fluorescence; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D010749:Phosphoprotein Phosphatases / Q000235:genetics* / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D000255:Adenosine Triphosphate; D017346:Protein Serine-Threonine Kinases; C042301:isocitrate dehydrogenase kinase; C042300:isocitrate dehydrogenase phosphatase; D010749:Phosphoprotein Phosphatases; D000251:Adenosine Triphosphatases