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Escherichia coli isocitrate dehydrogenase kinase/phosphatase. Overproduction and kinetics of interaction with its substrates by using intrinsic fluorescence and fluorescent nucleotide analogues.
Eur J Biochem. 1996 Apr 01; 237(1):247-54.EJ

Abstract

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.

Authors+Show Affiliations

Max-Planck-Institut für Medizinische Forschung, Abteilung Biophysik, Heidelberg, Germany.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

8620880

Citation

Rittinger, K, et al. "Escherichia Coli Isocitrate Dehydrogenase Kinase/phosphatase. Overproduction and Kinetics of Interaction With Its Substrates By Using Intrinsic Fluorescence and Fluorescent Nucleotide Analogues." European Journal of Biochemistry, vol. 237, no. 1, 1996, pp. 247-54.
Rittinger K, Negre D, Divita G, et al. Escherichia coli isocitrate dehydrogenase kinase/phosphatase. Overproduction and kinetics of interaction with its substrates by using intrinsic fluorescence and fluorescent nucleotide analogues. Eur J Biochem. 1996;237(1):247-54.
Rittinger, K., Negre, D., Divita, G., Scarabel, M., Bonod-Bidaud, C., Goody, R. S., Cozzone, A. J., & Cortay, J. C. (1996). Escherichia coli isocitrate dehydrogenase kinase/phosphatase. Overproduction and kinetics of interaction with its substrates by using intrinsic fluorescence and fluorescent nucleotide analogues. European Journal of Biochemistry, 237(1), 247-54.
Rittinger K, et al. Escherichia Coli Isocitrate Dehydrogenase Kinase/phosphatase. Overproduction and Kinetics of Interaction With Its Substrates By Using Intrinsic Fluorescence and Fluorescent Nucleotide Analogues. Eur J Biochem. 1996 Apr 1;237(1):247-54. PubMed PMID: 8620880.
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TY - JOUR T1 - Escherichia coli isocitrate dehydrogenase kinase/phosphatase. Overproduction and kinetics of interaction with its substrates by using intrinsic fluorescence and fluorescent nucleotide analogues. AU - Rittinger,K, AU - Negre,D, AU - Divita,G, AU - Scarabel,M, AU - Bonod-Bidaud,C, AU - Goody,R S, AU - Cozzone,A J, AU - Cortay,J C, PY - 1996/4/1/pubmed PY - 1996/4/1/medline PY - 1996/4/1/entrez SP - 247 EP - 54 JF - European journal of biochemistry JO - Eur J Biochem VL - 237 IS - 1 N2 - 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. SN - 0014-2956 UR - https://www.unboundmedicine.com/medline/citation/8620880/Escherichia_coli_isocitrate_dehydrogenase_kinase/phosphatase__Overproduction_and_kinetics_of_interaction_with_its_substrates_by_using_intrinsic_fluorescence_and_fluorescent_nucleotide_analogues_ L2 - https://onlinelibrary.wiley.com/resolve/openurl?genre=article&sid=nlm:pubmed&issn=0014-2956&date=1996&volume=237&issue=1&spage=247 DB - PRIME DP - Unbound Medicine ER -