In ( ), V 1 is the limiting rate of the primary reaction, and K M 1 is its Michaelis constant . The Michaelis constant is defined operationally as the concentration of the substrate at which the rate of the reaction is half of the limiting rate; that is, dp/dt = 0.5V 1 . The enzyme specificity is characterized by the specificity constant, which is the result of dividing k 2 by K M 1 . The kinetic constants (V 1 and K M 1 ) are generally estimated through initial rate or time course experiments by mathematically solving an inverse problem . In the case of coupled enzyme assays, the caveat with this procedure is that the primary reaction is not experimentally observable. Thus, V 1 and K M 1 need to be estimated through means of indirect measures of data recorded from the indicator reaction. From a theoretical point of view, the demand is obvious: a mathematical theory must be developed that is capable of accurately describing the relationship between the non-observable reaction and the indicator reaction. Most enzyme kinetic analyses developed to study coupled enzyme assays assume that the coupled enzyme reactions follow first-order kinetics . The limiting fact with this assumption is that first order models are typically only valid during the lag time of the reaction, which is effectively the length of time it takes before measurable formation rates of P become experimentally detectable. If the indicator reaction is fast, then first order models are only valid over very small timescales; this limits the duration of time over which useful (in the context of the inverse problem) experimental measurements can be made.