Determination of Michaelis-Menten and inhibitor constants by an open-closed flow injection approach (Application to the alkaline phosphatase/theophylline system)

An automatic method for the estimation of Michaelis-Menten and inhibitor constants in biochemical systems was developed using an unsegmented open-closed flow manifold, and the Lineweaver-Burk and Dixon plot methods. The methods were applied to both the determination of K(m) of the enzymatic reaction catalyzed by bovine alkaline phosphatase using 4-methylumbelliferone phosphate as substrate, and the determination of the K(i) constant from theophylline as an inhibitor of this biochemical system. The data treatment and comparative results are presented. The values obtained agree with those provided by a conventional method with the biocatalyst in solution.