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Class II recombinant phosphoribosyl diphosphate synthase from spinach. Phosphate independence and diphosphoryl donor
1Department of Biological Chemistry, Institute of Molecular Biology, University of Copenhagen, 83H Sølvgade, DK-1307 Copenhagen K, Denmark.
The Journal of Biological Chemistry
|March 30, 2001
Summary
Spinach phosphoribosyl diphosphate (PRPP) synthase isozyme 3 was synthesized and purified. This enzyme is P(i)-independent and shows unique substrate specificity and kinetics, differing from other PRPP synthases.
Area of Science:
- Biochemistry
- Enzymology
- Plant Science
Background:
- Phosphoribosyl diphosphate (PRPP) synthase is a crucial enzyme in nucleotide biosynthesis.
- Understanding PRPP synthase isozymes provides insights into metabolic regulation in plants.
Purpose of the Study:
- To characterize a recombinant spinach (Spinacia oleracea) PRPP synthase isozyme 3.
- To investigate its kinetic properties, substrate specificity, and cofactor requirements.
Main Methods:
- Recombinant expression of spinach PRPP synthase isozyme 3 in Escherichia coli.
- Enzyme purification to near homogeneity.
- Enzymatic assays to determine kinetic parameters and inhibitor effects.
Main Results:
- The purified enzyme exhibited P(i)-independent activity, unlike other PRPP synthases.
- Ribonucleoside diphosphates competitively inhibited the enzyme, indicating no allosteric inhibition.
- The enzyme accepted various diphosphoryl donors (dATP, GTP, CTP, UTP) besides ATP.
- Kinetic analysis revealed an ordered Bi Bi mechanism with specific K(m) and V(max) values.
- Optimal activity was observed at 40°C and pH 7.6, with an absolute requirement for magnesium ions.
Conclusions:
- Spinach PRPP synthase isozyme 3 possesses unique biochemical and kinetic properties.
- These characteristics suggest distinct regulatory mechanisms compared to PRPP synthases from other sources.
- The findings contribute to a deeper understanding of purine and pyrimidine metabolism in plants.
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