Kinetic studies of nitrogenase from soya-bean root-nodule bacteroids

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Nitrogenase is a crucial enzyme for nitrogen fixation in legumes.
• Understanding the kinetics of nitrogenase is essential for improving crop yields.

Purpose of the Study:

• To investigate the factors affecting the apparent Michaelis constants and maximum velocity of soya-bean bacteroid nitrogenase.
• To elucidate the role of nitrogenase Fe-protein and ATP in the enzyme's catalytic activity.

Main Methods:

• Cell-free assays were used to measure nitrogenase activity under varying conditions.
• Intact nodules and bacteroid suspensions were utilized to study the effects of O(2) pressure.
• Kinetic parameters, including apparent Michaelis constants (K'(N(2)), K'(C(2)H(2))) and apparent maximum velocity (V'), were determined.

Main Results:

• Apparent Michaelis constants and maximum velocity values increased with higher nitrogenase Fe-protein and ATP concentrations in cell-free assays.
• Oxygen pressure in intact nodules and bacteroid suspensions also correlated with increased kinetic parameters.
• pH and Na(2)S(2)O(4) concentration affected K'(C(2)H(2)) in cell-free assays.
• Nitrogenase Fe-protein acted as a catalytic effector on the nitrogenase Fe-Mo-protein, suggesting the Fe-Mo-protein contains the catalytic sites.

Conclusions:

• The Fe-Mo-protein is likely responsible for the catalytic reduction of N(2) and C(2)H(2).
• The reaction mechanism for N(2) or C(2)H(2) reduction by nitrogenase involves a sequence of unknown order.
• The interplay of substrate, enzyme, effector, ATP, and reductant influences the apparent Michaelis constant, indicating unresolved kinetic complexities.