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Dihydroorotase from Escherichia coli. Purification and characterization
The Journal of Biological Chemistry
|March 10, 1984
Summary
Dihydroorotase, an enzyme crucial for pyrimidine biosynthesis, was purified from E. coli. This study details its dimeric structure, zinc content, and kinetic properties, providing insights into its catalytic mechanism.
Area of Science:
- Biochemistry
- Enzymology
- Molecular Biology
Background:
- Dihydroorotase (EC 3.5.2.3) is a key enzyme in the de novo pyrimidine biosynthesis pathway.
- Understanding its properties is essential for metabolic studies and potential therapeutic targeting.
Purpose of the Study:
- To purify dihydroorotase to homogeneity from an over-producing Escherichia coli strain.
- To characterize the physical and kinetic properties of the purified enzyme.
Main Methods:
- Enzyme purification from E. coli cell paste.
- Determination of molecular weight using equilibrium sedimentation and SDS-PAGE.
- Analysis of subunit composition and zinc content.
- Isoelectric focusing for charge heterogeneity assessment.
- Enzymatic assays to determine kinetic parameters (Michaelis constant and kcat).
Main Results:
- Purification yielded approximately 7 mg of pure dihydroorotase with a 35% yield.
- Native molecular weight was ~80,900 Da, indicating a dimeric structure (subunit MW ~38,400-41,000 Da).
- The enzyme contains ~0.95 zinc atoms per subunit.
- Isoelectric focusing revealed two active species with pI values of 4.97 and 5.26.
- Kinetic parameters were determined for both substrates, dihydro-DL-orotate and N-carbamyl-DL-aspartate.
Conclusions:
- Dihydroorotase from E. coli is a zinc-containing dimer with distinct charge variants.
- The characterized kinetic parameters provide a basis for understanding its role in pyrimidine synthesis.