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Carbamoyl-phosphate synthase in Phycomyces blakesleeanus.

M J Alonso1, D De Arriaga, J Soler

  • 1Departamento de Bioquímica y Biología Molecular, Universidad de León, Spain.

Biochimica Et Biophysica Acta
|July 20, 1988
PubMed
Summary

Carbamoyl-phosphate synthase from Phycomyces blakesleeanus uses ammonia as an N donor and requires Mg2+. This enzyme, composed of two unequal subunits, is unstable and has optimal activity at pH 7.4-7.8.

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Area of Science:

  • Biochemistry
  • Enzymology
  • Fungal Metabolism

Background:

  • Carbamoyl-phosphate synthase is a key enzyme in nitrogen metabolism.
  • Understanding its properties in different organisms is crucial for metabolic pathway elucidation.

Purpose of the Study:

  • To purify and characterize carbamoyl-phosphate synthase from Phycomyces blakesleeanus.
  • To determine the enzyme's substrate specificity, kinetic properties, and stability.

Main Methods:

  • Enzyme purification from fungal mycelia.
  • Gel filtration for molecular weight estimation.
  • SDS-PAGE for subunit analysis.
  • Enzyme activity assays under varying conditions (pH, substrates, activators/inhibitors).

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Main Results:

  • Purified enzyme has a molecular weight of 188,000 Da, with subunits of 130,000 and 55,000 Da.
  • The enzyme utilizes ammonia (not L-glutamine) and requires Mg2+; N-acetyl-L-glutamate is not an activator.
  • Kinetic parameters determined: Km for ammonia (6.34 mM), bicarbonate (10.5 mM), and ATP·Mg2+ (0.93 mM).
  • Optimal pH is 7.4-7.8; transition temperature is 38.5°C; enzyme is inhibited by N-ethylmaleimide.

Conclusions:

  • The characterized carbamoyl-phosphate synthase from Phycomyces exhibits unique properties regarding its N donor and cofactor requirements.
  • The enzyme's instability and specific kinetic profile provide insights into its regulation and function in fungal biosynthesis.