Keeping the Distance: Activity Control in Solid-Supported Sucrose Phosphorylase by a Rigid α-Helical Linker of Tunable Spacer Length

Enzyme immobilization into carrier materials has broad importance in biotechnology, yet understanding the catalysis of enzymes bound to solid surfaces remains challenging. Here, we explore surface effects on the catalysis of sucrose phosphorylase through a fusion protein approach. We immobilize the enzyme via a structurally rigid α-helical linker [EA₃K] _<i>n</i> of tunable spacer length due to the variable number of pentapeptide repeats used (<i>n</i> = 6, 14, 19). Molecular modeling and simulation approaches delineate the conformational space sampled by each linker relative to its His-tag cap used for surface tethering. The population distribution of linker conformers gets broader, with a consequent shift of the enzyme-to-surface distance to larger values (≤15 nm), as the spacer length increases. Based on temperature kinetic studies, we obtain an energetic description of catalysis by the enzyme-to-linker fusions in solution and immobilize on Ni²⁺-chelate agarose. The solid-supported enzymes involve distinct changes in enthalpy-entropy partitioning within the frame of invariant Gibbs free energy of activation (Δ<i>G</i> ^‡ = ∼61 kJ/mol at 30 °C). The entropic contribution (-<i>T</i>Δ<i>S</i> ^‡) to Δ<i>G</i> ^‡ increases with the spacer length, from -16.4 kJ/mol in the linker-free enzyme to +7.9 kJ/mol in the [EA₃K]₁₉ linked fusion. The immobilized [EA₃K]₁₉ fusion protein is indistinguishable in its catalytic properties from the enzymes in solution, which behave identically regardless of their linker. Enzymes positioned closer to the surface arguably experience a higher degree of molecular organization ("rigidification") that must relax for catalysis through the additional uptake of heat, compensated by a gain in entropy. Increased thermostability of these enzymes (up to 2.8-fold) is consistent with the proposed rigidification effect. Collectively, our study reveals surface effects on the activation parameters of sucrose phosphorylase catalysis and shows their consistent dependence on the length of the surface-tethering linker. The fundamental insight here obtained, together with the successful extension of the principle to a different enzyme (nigerose phosphorylase), suggests that rigid linker-based control of the protein-surface distance can be used as an engineering strategy to optimize the activity characteristics of immobilized enzymes.