Estimating Kinetic Parameters when the Amount of Enzyme Added to an Assay Is Not a Controlled Variable

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Abstract
Reliable estimates of Michaelis constants (Km) and inhibitor constants may be obtained, in the absence of control over the amount of enzyme being added to any assay system, provided the following constraints are met. Michaelis-Menten kinetics are obeyed. Two rate measurements must be made with the same sample of enzyme: at low and high substrate concentration for determining Km or minus and plus an inhibitor for determining inhibitor constants. The Michaelis constant may be calculated from the equation: .**GRAPHIC**. Inhibitor constants are derived graphically from Lineweaver-Burk or Dixon plots, once the Km has been calculated. The above technique has been applied to study of the acetylene-reducing ability of intact legume plants [soybeans (Glycine max (L.) Merr.) or cowpeas (Vigna unguiculata (L.) Walp) with rhizobia]. The apparent Km for acetylene reduction by nitrogenase in legume nodules is .apprx. 1/100 atmosphere in the absence of N and .apprx. 1/40 atmosphere in its presence.