Purification and properties of porphobilinogen deaminase from Arabidopsis thaliana
Related Experiment Videos
1School of Biological Sciences, Queen Mary and Westfield College, University of London, U.K.
Abstract:
Porphobilinogen deaminase (EC 4.3.1.8) has been purified to homogeneity (16,000-fold) from the plant Arabidopsis thaliana in yields of 8%. The deaminase is a monomer of M(r) 35,000, as shown by SDS/PAGE, and 31,000, using gel-filtration chromatography. The pure enzyme has a Vmax. of 4.5 mumol/h per mg and a Km of 17 +/- 4 microM. Determination of the pI and pH optimum revealed values of 5.2 and 8.0 respectively. The sequence of the N-terminus was found to be NH2-XVAVEQKTRTAI. The deaminase is heat-stable up to 70 degrees C and is inhibited by NH3 and hydroxylamine. The enzyme is inactivated by arginine-, histidine- and lysine-specific reagents. Incubation with the substrate analogue and suicide inhibitor, 2-bromoporphobilinogen, results in chain termination and in inactivation.
Insights
Researchers purified plant porphobilinogen deaminase (PBG deaminase) from Arabidopsis thaliana. This key enzyme in plant tetrapyrrole biosynthesis is monomeric, heat-stable, and sensitive to specific inhibitors.
Area of Science:
- Biochemistry
- Plant Molecular Biology
- Enzymology
Background:
- Porphobilinogen deaminase (PBG deaminase) is crucial for tetrapyrrole biosynthesis in plants.
- Understanding plant enzyme kinetics and properties is vital for agricultural and biotechnological applications.
Purpose of the Study:
- To purify and characterize porphobilinogen deaminase from the model plant Arabidopsis thaliana.
- To determine the enzyme's kinetic parameters, physical properties, and inhibition characteristics.
Main Methods:
- Homogeneous purification of PBG deaminase using established biochemical techniques.
- Enzyme characterization including SDS-PAGE, gel filtration, Vmax, Km, pI, and pH optimum determination.
- Investigation of enzyme stability, inhibition by ammonia and hydroxylamine, and inactivation by specific reagents and a substrate analogue.
Main Results:
- PBG deaminase was purified 16,000-fold with 8% yield.
- The enzyme is a monomer with a molecular weight of approximately 35,000 Da.
- Kinetic analysis revealed a Km of 17 ± 4 μM and Vmax of 4.5 μmol/h/mg, with a pI of 5.2 and pH optimum of 8.0.
- The enzyme demonstrated heat stability up to 70°C and was inhibited by ammonia, hydroxylamine, and specific amino acid modifying reagents, as well as a suicide inhibitor.
Conclusions:
- The study provides a comprehensive biochemical characterization of Arabidopsis thaliana porphobilinogen deaminase.
- The enzyme's properties suggest specific structural and functional attributes relevant to tetrapyrrole synthesis pathways in plants.