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

NITROGENASE ACTIVITY OF INTACT LEGUMES ¹, ^,2

Department of Biochemistry, Kansas State University, Manhattan, Kansas 66506

Reliable estimates of Michaelis constants (K_m) 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 K_m or minus and plus an inhibitor for determining inhibitor constants. The Michaelis constant may be calculated from the equation [Formula: see text] Inhibitor constants are derived graphically from Lineweaver-Burk or Dixon plots, once the K_m has been calculated. The above technique has been applied to study of the acetylene-reducing ability of intact legume plants. The apparent K_m for acetylene reduction by nitrogenase in legume nodules is 1/100 atmosphere in the absence of nitrogen and 1/40 atmosphere in its presence.

² Contribution No. 80-196-j of the Kansas Agricultural Experiment Station.