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Related Experiment Videos

Nucleoside diphosphate kinase from Escherichia coli

N Almaula1, Q Lu, J Delgado

  • 1Department of Biochemistry, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA.

Journal of Bacteriology
|May 1, 1995
PubMed
Summary

Nucleoside diphosphate (NDP) kinase from E. coli is a tetramer. Phosphorylation occurs at histidine and serine residues, with Ser-119 and Ser-121 identified as key sites, though other minor sites exist.

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Area of Science:

  • Biochemistry
  • Enzymology
  • Protein Chemistry

Background:

  • Nucleoside diphosphate (NDP) kinase is crucial for cellular energy metabolism.
  • Understanding the enzyme's catalytic mechanism, including autophosphorylation, is key to its function.

Purpose of the Study:

  • To purify and characterize NDP kinase from Escherichia coli.
  • To identify the specific amino acid residues involved in the enzyme's autophosphorylation.
  • To investigate the role of identified residues in enzyme activity and phosphorylation.

Main Methods:

  • Purification of NDP kinase to homogeneity.
  • Crystallization and gel filtration analysis to determine enzyme structure.
  • Enzyme phosphorylation using [gamma-32P]ATP and pH stability profiling.

Related Experiment Videos

  • Site-directed mutagenesis (Ser119Ala, Ser121Ala, and double mutant) to assess residue function.

    • Main Results:

    • Purified E. coli NDP kinase forms a tetramer.
    • Phosphorylation occurs at both histidine and serine residues (Ser-119, Ser-121).
    • Mutant enzymes retained NDP kinase activity, and Ser119Ala/Ser121Ala double mutant showed minor, acid-resistant autophosphorylation, suggesting additional sites.

    Conclusions:

    • Identified key serine residues (Ser-119, Ser-121) in E. coli NDP kinase autophosphorylation.
    • Autophosphorylation mechanism is complex, involving multiple sites, including minor ones.
    • Findings provide insights into NDP kinase function and potential regulation, with implications for human NDP kinase studies.