Keeping the Distance: Activity Control in Solid-Supported\nSucrose Phosphorylase by a Rigid α‑Helical Linker of\nTunable Spacer Length

Enzyme immobilization\ninto carrier materials has broad importance\nin biotechnology, yet understanding the catalysis of enzymes bound\nto solid surfaces remains challenging. Here, we explore surface effects\non the catalysis of sucrose phosphorylase through a fusion protein\napproach. We immobilize the enzyme via a structurally rigid α-helical\nlinker [EA<sub>3</sub>K]<sub><i>n</i></sub> of tunable spacer\nlength due to the variable number of pentapeptide repeats used (<i>n</i> = 6, 14, 19). Molecular modeling and simulation approaches\ndelineate the conformational space sampled by each linker relative\nto its His-tag cap used for surface tethering. The population distribution\nof linker conformers gets broader, with a consequent shift of the\nenzyme-to-surface distance to larger values (≤15 nm), as the\nspacer length increases. Based on temperature kinetic studies, we\nobtain an energetic description of catalysis by the enzyme-to-linker\nfusions in solution and immobilize on Ni<sup>2+</sup>-chelate agarose.\nThe solid-supported enzymes involve distinct changes in enthalpy–entropy\npartitioning within the frame of invariant Gibbs free energy of activation\n(Δ<i>G</i><sup>‡</sup> = ∼61 kJ/mol\nat 30 °C). The entropic contribution (−<i>T</i>Δ<i>S</i><sup>‡</sup>) to Δ<i>G</i><sup>‡</sup> increases with the spacer length, from −16.4\nkJ/mol in the linker-free enzyme to +7.9 kJ/mol in the [EA<sub>3</sub>K]<sub>19</sub> linked fusion. The immobilized [EA<sub>3</sub>K]<sub>19</sub> fusion protein is indistinguishable in its catalytic properties\nfrom the enzymes in solution, which behave identically regardless\nof their linker. Enzymes positioned closer to the surface arguably\nexperience a higher degree of molecular organization (“rigidification”)\nthat must relax for catalysis through the additional uptake of heat,\ncompensated by a gain in entropy. Increased thermostability of these\nenzymes (up to 2.8-fold) is consistent with the proposed rigidification\neffect. Collectively, our study reveals surface effects on the activation\nparameters of sucrose phosphorylase catalysis and shows their consistent\ndependence on the length of the surface-tethering linker. The fundamental\ninsight here obtained, together with the successful extension of the\nprinciple to a different enzyme (nigerose phosphorylase), suggests\nthat rigid linker-based control of the protein–surface distance\ncan be used as an engineering strategy to optimize the activity characteristics\nof immobilized enzymes.