Both enzyme reactions obey a Michaelis-Menten (MM) mechanism of enzyme action . Experimental scientists assume that the product is released irreversibly in enzyme catalyzed reactions. This assumption can be safely made for coupled enzyme assays because reactions are chosen on the basis of thermodynamic properties that make them nearly irreversible . Coupled (or auxiliary) enzyme reactions generally exhibit a lag or transient time , which is effectively the length of time it takes before the rate of formation of P reaches its maximum value. From the chemical kinetics point of view, the lag time is essentially the amount of time it takes (2) to reach the quasi-steady-state (QSS) period and the initialization of product synthesis . Therefore, when the quasi-steady-state assumption (QSSA) is applied to the coupled (or auxiliary) enzyme reaction (1)-( ), the lag time coincides with the initial transient timescale over which the intermediate complex C 2 is accumulating to its threshold value . Most of the theory concerning coupled (or auxiliary) enzyme reactions assays has concentrated on the derivation of mathematical expressions to estimate the lag time . While the rate of formation of P is generally small prior to a significant accumulation in C 2 , previous theoretical work assumed that the onset of the QSS always corresponds to onset of C 2 having reached its critical maximum value. This is because the steady-state production of P does not occur until C 2 reaches this critical threshold value. To derive lag time expressions, numerous assumptions have been made about the dynamical behavior of coupled enzyme assays. Most work has assumed that coupled enzyme catalyzed reactions follow first-order kinetics ; this assumption limits the validity of the lag time expressions and practical usage of the time course expressions of the reactions . Other studies have relied on using single-substrate, single-enzyme MM equations for the non-observable and indicator reactions, but these expressions are simplified to pseudo-first order equations by assuming that the enzyme assays are performed at substrate concentrations lower than their Michaelis constants . showed that MM equations for uncoupled reactions can adequately describe coupled (or auxiliary) enzyme assays if the three conditions are met: (i) the rate of the non-observable enzyme is constant, (ii) the coupled enzyme reactions are irreversible, and (iii) the indicator reaction has a short-lived intermediate with a concentration smaller or equal to its Michaelis constant.