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

Abstract

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, non-signaling 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 non-signaling 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.

Using Distal-Site Mutations and Allosteric Inhibition To Tune, Extend, and Narrow the Useful Dynamic Range of Aptamer-Based Sensors

“…This fixed dynamic range limits the usefulness of such receptors in applications requiring the measurement of target concentration over many orders of magnitude. Other applications, such as molecular logic gates, biomolecular systems programmed to integrate multiple inputs (i.e., multiple disease biomarkers) into a single output 3 , could likewise benefit from strategies that provide steeper, more “digital” input-output response curves 4 .…”

Immobilization of enzymes to silver island films for enhanced enzymatic activity

“…[1] For example, in biosensors, enzymes are employed as recognition and signaling elements for the detection of specific molecular analyte of interest. [2], [3], [4] In this regard, enzymes are immobilized on to surfaces through covalent binding [4], direct crosslinking, [5] and encapsulation [6] of enzymes on different platforms such as alumina, [7] silica, [8] electrode [4] and nanoparticles. [9] The extent of enzymatic activity after surface immobilization depends on the binding procedure and on the availability of enzymes to substrates.…”

Glucose oxidase-encapsulated nanogold hollow microspheres as labels based on a sensitive electroluminescent immunoassay

“…[22][23][24] To date, enzymes have been widely applied as recognition and signaling elements for the detection of some specic molecular analytes. Furthermore, the ECL strategy can decrease the detection limit to the single-molecular level.…”