Fig. 3: Controlling and monitoring the rate and directionality of an enzyme cascade.

a The nanoconfined cascade in downstream notation. Enzymes E1 and E2 are, respectively, FNR and a NAD(P)(H) dehydrogenase and they represent the transducer-engine pair at which the rate of reaction flow along the cascade is recorded directly as current. Here the rest of the cascade is downstream with respect to the cofactor-dependent step (E1–E2 pair). Intermediates are passed to the next enzyme or escape (dotted red lines) into the bulk solution. b The nanoconfined cascade in upstream notation: the rest of the cascade is upstream of the cofactor-dependent step. c Rotation-rate dependence experiment for the system (CA/FNR/ME/FumC/AspA)@ITO/PGE (0.03 cm²) in a buffer containing NH₄Cl, KHCO₃, and pyruvate at pH 7.5, 25 °C, rotation rate 1000 rpm, under N₂ atmosphere. Arrows signify injection of NADP⁺ (to a final concentration of 20 µM) and switching rotation off (0 rpm) and on (1000 rpm). d Continuation of the experiment carried out in panel c: the cell was washed carefully, a fresh buffer was added and rotation (1000 rpm) was switched on. Arrows signify injection of NADP⁺ (to 20 µM) and aspartate (substrate of E4, to 20 mM) and successive switching off and on of rotation. e Cascade with E1, E2, etc, identified with the different enzymes making up the cascade unit: downstream direction runs left to right.