Michaelis constants of non-chromogenic substrates may be determined using chromogenic substrates

The object of these experiments was to demonstrate a means of determining the Michaelis constants (Km and Vmax) of a non-chromogenic substrate in a three-part experimental system containing a non-chromogenic substrate, the corresponding enzyme, and an appropriate chromogenic substrate. Two experimental systems were examined, one in which the enzyme activity was subject to product inhibition (alkaline phosphatase) and one in which the activity was not (..beta..-lactamase). In both systems, the initial experimental goal was to quantify the area defined by the entire kinetic trace (absorbance versus time) obtained with the chromogenic substrate in the absence or presence of the non-chromogenic substrate. The difference between these two areas was directly proportional to the concentration of the non-chromogenic substrate and inversely proportional to the Vmax of the non-chromogenic substrate. The value of Vmax thus obtained could then be used with the appropriate integrated Michaelis-Menten rate equation to extract the value of the Km for the non-chromogenic substrate. This method yielded values of kinetic constants that were in good agreement with those obtained directly and/or those available from the literature. It is anticipated that this method may be extended to enzymes with more complicated kinetic mechanisms.