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

Abstract

The apparent Michaelis constants [K′(N₂) and K′(C₂H₂)] and the corresponding apparent maximum velocity values (V′) for soya-bean bacteroid nitrogenase increased concomitantly in response to increases in nitrogenase Fe–protein concentration and ATP concentration in cell-free assays and in response to O₂ pressure in intact nodules and bacteroid suspensions. K′(C₂H₂) in cell-free assays was also affected by pH and by Na₂S₂O₄ concentration. Nitrogenase Fe–protein behaved as a catalytic effector reacting at interacting sites on the nitrogenase Fe–Mo–protein. The results indicated that the Fe–Mo–protein probably bears the catalytic sites for N₂ and C₂H₂ reduction. It is concluded that reduction of N₂ or C₂H₂ by this nitrogenase involves a reaction mechanism with a sequence of unknown order. The sequence in which substrate, enzyme, effector, ATP and reductant react determines which of the various rate-constants are involved in the apparent Michaelis constant, whose true kinetic meaning was thus unresolved.