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Purification and characterization of dichloromuconate cycloisomerase from Alcaligenes eutrophus JMP 134
A E Kuhm1, M Schlömann, H J Knackmuss
1Institut für Mikrobiologie, Universität Stuttgart, Federal Republic of Germany.
Abstract:
Dichloromuconate cycloisomerase from Alcaligenes eutrophus JMP 134 was purified to homogeneity. The enzyme has an Mr of about 270,000 as determined by gel filtration and consists of six to eight subunits of identical Mr 40,000 as determined by SDS/PAGE. Mn2+ ions as well as thiol groups are required for activity. A high Km value of about 4 mM for cis,cis-muconate explains the reported low activity with this compound. Relatively high Km values were also calculated for monochloro-substituted cis,cis-muconates (300-500 microM), in contrast with the low Km value of 20 microM for 2,4-dichloro-cis,cis-muconate. The catalytic constant of the pure enzyme was 3820 min-1 when measured with 2,4-dichloro-cis,cis-muconate.
Insights
Dichloromuconate cycloisomerase enzyme activity was characterized. The purified enzyme shows high specificity for 2,4-dichloro-cis,cis-muconate, indicating its role in chlorinated aromatic compound degradation.
Area of Science:
- Biochemistry
- Enzymology
- Environmental Microbiology
Background:
- Dichloromuconate cycloisomerase is involved in the degradation of chlorinated aromatic compounds.
- Understanding its enzymatic properties is crucial for bioremediation strategies.
Purpose of the Study:
- To purify and characterize dichloromuconate cycloisomerase from Alcaligenes eutrophus JMP 134.
- To determine the enzyme's kinetic parameters and cofactor requirements.
Main Methods:
- Enzyme purification to homogeneity using gel filtration and SDS/PAGE.
- Enzyme activity assays with various muconate substrates.
- Determination of kinetic parameters (Km, catalytic constant) and cofactor dependency (Mn2+, thiol groups).
Main Results:
- Purified enzyme has a molecular mass of approximately 270,000 Da, composed of 6-8 subunits.
- Enzyme activity requires Mn2+ ions and thiol groups.
- High Km for cis,cis-muconate (4 mM) and monochloro-muconates (300-500 microM), but low Km for 2,4-dichloro-cis,cis-muconate (20 microM).
- Catalytic constant for 2,4-dichloro-cis,cis-muconate is 3820 min-1.
Conclusions:
- Dichloromuconate cycloisomerase exhibits substrate specificity towards chlorinated muconates.
- The enzyme's kinetic properties suggest a specialized role in the metabolism of 2,4-dichloromuconate.
- Findings contribute to understanding microbial degradation pathways of pollutants.