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Here, we demonstrate a strategy to convert the graded Michaelis-Menten response typical of unregulated enzymes into a sharp, effectively all-or-none response. We do so using an approach analogous to the "branch point effect", a mechanism observed in naturally occurring metabolic networks in which two or more enzymes compete for the same substrate. As a model system, we used the enzymatic reaction of glucose oxidase (GOx) and coupled it to a second, nonsignaling reaction catalyzed by the higher affinity enzyme hexokinase (HK) such that, at low substrate concentrations, the second enzyme outcompetes the first, turning off the latter's response. Above an arbitrarily selected "threshold" substrate concentration, the nonsignaling HK enzyme saturates leading to a "sudden" activation of the first signaling GOx enzyme and a far steeper dose-response curve than that observed for simple Michaelis-Menten kinetics. Using the well-known GOx-based amperometric glucose sensor to validate our strategy, we have steepen the normally graded response of this enzymatic sensor into a discrete yes/no output similar to that of a multimeric cooperative enzyme with a Hill coefficient above 13. We have also shown that, by controlling the HK reaction we can precisely tune the threshold target concentration at which we observe the enzyme output. Finally, we demonstrate the utility of this strategy for achieving effective noise attenuation in enzyme logic gates. In addition to supporting the development of biosensors with digital-like output, we envisage that the use of all-or-none enzymatic responses will also improve our ability to engineer efficient enzyme-based catalysis reactions in synthetic biology applications.

Employing the Metabolic “Branch Point Effect” to Generate an All-or-None, Digital-like Response in Enzymatic Outputs and Enzyme-Based Sensors