The simulated concentrations were rate constant values were k sub = 0.00001 µM -1 s -1 , k on = 0.1 µM -1 s -1 , and k off = 0.0001 s -1 . Thus the equilibrium dissociation constant of the enzyme-inhibitor complex K i = k off /k on = 1 nM, which is ten times lower than the enzyme concentration. Also note that approximately one half of the inhibitor concentrations are lower than the enzyme concentration (tightbinding). Altogether 60 data points were simulated for each progress curve spanning from time zero to t = 3600 s, or one hour. The normally distributed random experimental noise added to each data point had standard deviation equal to 0.7 percent of the maximum signal. The instantaneous reaction rates associated with the simulated progress curves are shown in Figure . Note that, importantly, the reaction rate simulated at zero inhibitor concentration is unchanging, which means that the reaction progress curve simulated in the absence of inhibitors is strictly linear. This fully justifies the assumptions inherent in the integrated rate equation Eqn . Also note that at least several of the relatively high inhibitor concentration curves did reach a steady-state rate, which means that the simulated data set should contain sufficient inhibition to determine the reversible binding affinity.