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Ribulose diphosphate carboxylase from autotrophic microorganisms.
Journal of Bacteriology
|May 1, 1972
Summary
This study investigated ribulose diphosphate carboxylase in Thiobacillus denitrificans, finding it stable during growth and essential for CO2 fixation. Enzyme activity was dependent on bicarbonate and Mg2+, with cyanide causing significant inhibition.
Area of Science:
- Microbial physiology
- Enzymology
- Biochemistry
Background:
- Thiobacillus denitrificans utilizes nitrate as an electron acceptor under anaerobic conditions.
- Ribulose diphosphate carboxylase (RuBisCO) is a key enzyme in carbon fixation pathways.
- Understanding RuBisCO function in diverse microorganisms is crucial for microbial ecology and biotechnology.
Purpose of the Study:
- To characterize the stability and activity of ribulose diphosphate carboxylase in Thiobacillus denitrificans.
- To investigate the kinetic properties and cofactor dependencies of the purified enzyme.
- To compare the T. denitrificans enzyme with RuBisCO from other organisms.
Main Methods:
- Anaerobic growth of Thiobacillus denitrificans in sterile and nonsterile media.
- Enzyme purification using standard biochemical techniques, achieving 50-60% purity.
- Enzyme activity assays measuring CO2 fixation rates under varying conditions (pH, substrate concentrations).
- Kinetic analysis of enzyme dependence on bicarbonate, ribulose diphosphate, and Mg2+.
- Cross-reactivity studies using antiserum and inhibition assays with cyanide.
Main Results:
- Ribulose diphosphate carboxylase remained stable throughout the exponential growth phase of T. denitrificans.
- Purified enzyme exhibited a molecular weight of approximately 350,000 daltons and catalyzed CO2 fixation at 1.25 µmol/min/mg protein.
- Enzyme activity showed biphasic kinetics, broad pH optimum (7-8.2), and required pre-incubation with bicarbonate and Mg2+.
- Cyanide strongly inhibited the T. denitrificans enzyme (61%), while antiserum from H. eutropha showed minimal inhibition.
Conclusions:
- Ribulose diphosphate carboxylase in T. denitrificans is relatively stable and plays a vital role in carbon assimilation.
- The enzyme's kinetic properties suggest a specific order of substrate binding.
- Enzyme structure and function may vary across different bacterial species, as indicated by limited cross-reactivity and differential cyanide sensitivity.