The michaelis constants of a nonchromogenic substrate may be determined using a chromogenic substrate

Abstract A general method is presented for the determination of the K M and V max for a nonchromogenic substrate in an experimental system where a chromogenic and a nonchromogenic substrate compete for the active site of a single enzyme. Entire progress curves of absorbance versus time for the transformation of the chromogenic substrate must be obtained in the absence and presence of the nonchromogenic substrate. Two quantities may then be extracted from the entire kinetic curves: the value of ΔArea, the area bounded by the kinetic traces of absorbance versus time in the presence and absence of the nonchromogenic substrate; and the value of Δt (5%), the time required to transform 5% of the chromogenic substrate in the presence of the nonchromogenic substrate minus the corresponding time required in its absence. The values of K M and V max for the nonchromogenic substrate may be obtained from the dependencies of ΔArea and Δt (5%) upon the concentration of the nonchromogenic substrate. The ability of this procedure to yield the correct values of K M and V max was demonstrated using β-lactamase, β-galactosidase, alkaline phosphatase, and 19 chromogenic/nonchromogenic substrate pairs. This method is equally valid in the absence or presence of competitive product inhibition and should be applicable to any enzyme-catalyzed, strongly exergonic reaction.