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Related Experiment Videos

Studies on site directed mutant pig citrate synthases.

C T Evans1, D Owens, J P Casazza

  • 1Department of Veterans Affairs, Dallas, Texas.

Biochemical and Biophysical Research Communications
|November 15, 1989
PubMed
Summary

Key amino acids His274 and Asp375 are crucial for pig citrate synthase (PCS) enzyme activity. Mutations in these residues prevent PCS function in E. coli, highlighting their essential role in catalysis.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Enzymology

Background:

  • Citrate synthase is a vital enzyme in the citric acid cycle.
  • Understanding enzyme active sites is crucial for metabolic pathway analysis.
  • Bacterial citrate synthase deficient strains (gltA-) provide a model for studying heterologous enzyme function.

Purpose of the Study:

  • To investigate the role of specific amino acids (His274 and Asp375) in the catalytic activity of porcine citrate synthase (PCS).
  • To assess the synthesis and stability of non-mutant and mutant PCS in a bacterial expression system.
  • To determine the functional consequences of mutations at key active site residues.

Main Methods:

  • Subcloning of non-mutant and mutant PCS DNA into an E. coli expression system.

Related Experiment Videos

  • Growth assays of transformed E. coli gltA- cells on minimal acetate media.
  • Protein blot and immunoisolation analyses to confirm protein presence.
  • Enzyme activity assays on cell lysates.

    • Main Results:

    • Non-mutant PCS expression restored growth in E. coli gltA- cells.
    • Mutations at His274 or Asp375 abolished PCS activity and prevented cell growth.
    • Mutant PCS proteins were synthesized but exhibited significantly reduced enzyme activity (two orders of magnitude lower).

    Conclusions:

    • Histidine 274 and Aspartate 375 are essential active site residues for porcine citrate synthase catalysis.
    • These findings underscore the critical role of specific amino acids in enzyme function.
    • The study validates the use of E. coli gltA- mutants for studying citrate synthase activity.