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Related Experiment Videos

Substrate binding domains in pyruvate phosphate dikinase

L J Carroll1, Y Xu, S H Thrall

  • 1Department of Chemistry and Biochemistry, University of Maryland, College Park 20742.

Biochemistry
|February 8, 1994
PubMed
Summary

Subtilisin Carlsberg proteolysis of pyruvate phosphate dikinase (PPDK) reveals distinct cleavage pathways. These studies suggest PPDK has multiple structural domains, with separate sites for catalysis, pyruvate, and ATP.

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Area of Science:

  • Enzymology
  • Protein Chemistry
  • Structural Biology

Background:

  • Pyruvate phosphate dikinase (PPDK) is a crucial enzyme in gluconeogenesis.
  • Understanding PPDK's structure is key to elucidating its catalytic mechanism.

Purpose of the Study:

  • To investigate the structural domains of Clostridium symbiosum PPDK using limited proteolysis.
  • To map potential binding sites for substrates and cofactors within PPDK.

Main Methods:

  • Limited proteolysis of PPDK using subtilisin Carlsberg.
  • Analysis of proteolytic fragments by SDS-PAGE.
  • Radiolabeling studies with bromopyruvate and adenosine 5'-monophosphate analogs.

Main Results:

  • Two major proteolytic cleavage pathways were identified for free and phosphorylated PPDK.

Related Experiment Videos

  • Oxalate binding induced a conformational change, altering proteolysis patterns.
  • Specific fragments were selectively radiolabeled, indicating distinct binding sites.

    • Conclusions:

    • PPDK possesses multiple structural domains.
    • The catalytic histidine, pyruvate, and ATP binding sites are likely located on different domains.
    • Limited proteolysis is a valuable tool for probing enzyme structure and function.